Camptothecin analogs and methods of preparation thereof

ABSTRACT

A compound and a method of synthesizing a compound having the following general formula (1): ##STR1## wherein R 1  and R 2  are independently the same or different and are hydrogen, an alkyl group, an alkenyl group, a benzyl group, an alkynyl group, an alkoxy group, an aryloxy group, an acyloxy group, --OC(O)OR d , wherein R d  is an alkyl group, a carbamoyloxy group, a halogen, a hydroxy group, a nitro group, a cyano group, an azido group, a formyl group, a hydrazino group, an acyl group, an amino group, --SR c , wherein, R c  is hydrogen, an acyl group, an alkyl group, or an aryl group, or R 1  and R 2  together form a group of the formula --O(CH 2 ) n  O-- wherein n represents the integer 1 or 2; R 3  is H, F, a halogen atom, a nitro group, an amino group, a hydroxy group, or a cyano group; or R 2  and R 3  together form a group of the formula --O(CH 2 ) n  O-- wherein n represents the integer 1 or 2; R 4  is H, a trialkylsilyl group, F, a C 1-3  alkyl group, a C 2-3  alkenyl group, a C 2-3  alkynyl group, or a C 1-3  alkoxy group; R 5  is a C 1-10  alkyl group, an allyl group, a benzyl group or a propargyl group; and R 6 , R 7  and R 8  are independently a C 1-10  alkyl group, a C 2-10  alkenyl group, a C 2-10  alkynyl group, an aryl group or a --(CH 2 ) N  R 9  group, wherein N is an integer within the range of 1 through 10 and R 9  is a hydroxy group, alkoxy group, an amino group, an alkylamino group, a dialkylamino group, a halogen atom, a cyano group or a nitro group; and R 11  is an alkylene group or an alkenylene group, and pharmaceutically acceptable salts thereof.

GOVERNMENT INTERESTS

This invetnion was made with government support under grant #5 RO1GM031678 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 08/921,102, filed Aug. 29, 1997, which acontinuation-in-part application of U.S. patent application Ser. No.08/436,799 filed May 8, 1995, now abandoned, which is acontinuation-in-part application of U.S. patent application Ser. No.08/085,190 filed Jun. 30, 1993, now abandoned, the disclosures of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel compounds and methods ofpreparation thereof and, particularly, to silyl camptothecin derivativesor analogs and to methods of preparation of such silyl camptothecinanalogs.

BACKGROUND OF THE INVENTION

(20S)-Camptothecin (CPT, see below) and its derivatives are some of themost promising agents for the treatment of solid tumors by chemotherapy.See, for example, Wall, M. E. et al, J. Ethnopharmacol., 51, 239 (1996);Camptothecin: New Anticancer Agents; Potmesil, M. and Pinedo, H., Eds.;CRC, Boca Raton, Fla. (1995); Bonneterre, J., Bull. Canc., 82, 623(1995); Sinha, D. K., Drugs, 49, 11 (1995). This natural alkaloid wasfirst isolated in 1966 from the extract of a Chinese plant, Camptothecaaccuminata, by Wall. Wall, M. E. et al, J. Am. Chem. Soc., 88, 3888(1966). As depicted below, camptothecin has a fused ring systemgenerally comprising a pyrrolo[3,4-b]quinoline system (rings ABC) fusedto a 2-pyridone ring (ring D), which, in turn, is fused to a lactonering (ring E). ##STR2##

Camptothecin belongs to the family of topoisomerase I poisons. See, forexample, Froelich-Ammon, S. J. et al., J. Biol. Chem., 270, 21429(1995). Research to date strongly suggests that this molecule acts byinterfering with the unwinding of supercoiled DNA by the cellular enzymetopoisomerase I, an enzyme which is usually overexpressed in malignantcells. In the highly replicating cancer cells, this triggers a cascadeof events leading to apoptosis and programmed death. See Slichenmyer, W.J. et al., J. Natl. Cancer Inst., 85, 271 (1993). Recent advances at themolecular pharmacology level are reviewed in Pommier, Y. et al., Proc.Natl. Acad. Sci. USA, 92, 8861 (1995).

Camptothecin's initial clinical trials were limited by its poorsolubility in physiologically compatible media. Moreover, early attemptsto form a water-soluble sodium salt of camptothecin by opening thelactone ring with sodium hydroxide resulted in a compound having a poorantitumor activity. It was later reported that the closed lactone-formis an absolute requisite for antitumor activity. See Wani, M. C. et al.,J. Med. Chem., 23, 554 (1980). More recently, structure-activity studieshave identified analogous compounds with better solubility and betterantitumor activity. For example, topotecan (TPT) and irinotecan (IRT)have recently been approved for sale in the United States, whileGI-147211C is in late stage clinical trials. These analogs are effectiveagainst a variety of refractory solid tumors such as malignant melanoma,stomach, breast, ovarian, lung and colorectal cancers, and seemparticularly promising for the treatment of slow-dividing cancer lines.See, for example, Kingsbury, W. D. et al., J. Med. Chem., 34, 98 (1991);Sawada, S. et al., Chem. Pharm. Bull., 39, 1446 (1991); Luzzio, M. J. etal., J. Med. Chem., 38, 395 (1995); Abigerges, D. et al., J. Clin.Oncol., 13, 210 (1995). Furthermore, synergistic or additive effectshave been observed in combination therapies with cisplatin, irradiation,or hyperthermia. See Fukuda, M. et al., Canc. Res., 56, 789 (1996);Goldwasser, F. et al., Clin. Canc. Res., 2, 687 (1996); Wang, D. S. etal., Biol. Pharm. Bull., 19, 354 (1996).

Although most research has focused on the development of water-solublederivatives of camptothecin, new formulations, such aslipid-complexation, liposomal encapsulation, and wet milling technologyhave recently been developed. Such formulations result in newtherapeutic opportunities for poorly water-soluble camptothecins. SeeDaoud, S. S. et al., Anti-Cancer Drugs, 6, 83 (1995);Merisko-Liversidge, E. et al., Pharm. Res., 13, 272 (1996); andPantazis, P., Leukemia Res., 19, 775 (1995). An attractive feature ofthese formulations is their impact on drug biodistribution. Sugarman andcoworkers have recently reported that while free camptothecin achievesthe greatest concentration in the pulmonary parenchyma, lipid-complexedcamptothecin has the highest concentration in the gastrointestinaltract. These results open new and interesting perspectives for thetreatment of colon cancer. See Sugarman, S. M. et al., Canc. Chemother.Pharmacol., 37, 531 (1996). Another interesting aspect of usinginsoluble camptothecin analogs is that they are usually more active thantheir water-soluble congeners and seem less likely to createdrug-induced resistance, probably because they are not substrates of thep-glycoprotein multi-drug transporter. See Pantazis, P., Clin. Canc.Res., 1, 1235 (1995).

In this context, new camptothecin analogs that combine good to excellentanti-tumor activities with different solubility and biodistributionprofiles could play a crucial role in the therapeutic arsenal for thetreatment of various types of cancers.

Given the proven beneficial biological activity of camptothecin andanalogs thereof, it is desirable to develop additional camptothecinanalogs and methods of preparation of camptothecin analogs.

SUMMARY OF THE INVENTION

The present invention provides generally a compound having the followingformula (1): ##STR3##

The present invention also provides a method of synthesizing compoundshaving the formula (2): ##STR4## via a 4+1 radicalannulation/cyclization wherein the precursor ##STR5## is reacted with anaryl isonitrile having the formula ##STR6##

R¹ and R² are independently the same or different and are preferablyhydrogen, an alkyl group, an alkenyl group, an alkynyl group, an alkoxygroup, an aryloxy group, an acyloxy group, --OC(O)OR^(d), wherein R^(d)is an alkyl group, a carbamoyloxy group, a halogen, a hydroxy group, anitro group, a cyano group, an azido group, a formyl group, a hydrazinogroup, an acyl group (--C(O)R^(f) wherein R^(f) is preferably an alkylgroup, an alkoxy group, an amino group or a hydroxy group), an aminogroup, --SR^(c), wherein, R^(c) is hydrogen, an acyl group, an alkylgroup, or an aryl group, or R¹ and R² together form a group of theformula --O(CH₂)_(n) O-- wherein n represents the integer 1 or 2.

R³ is preferably H, a halogen, a nitro group, an amino group, a hydroxygroup, or a cyano group. R² and R³ can also together form a group of theformula --O(CH₂)_(n) O-- wherein n represents the integer 1 or 2.

R⁴ is preferably H, F, a trialkylsilyl group, a C₁₋₃ alkyl group, a C₂₋₃alkenyl group, a C₂₋₃ alkynyl group, or a C₁₋₃ alkoxy group. R⁵ ispreferably a C₁₋₁₀ alkyl group. A preferred alkyl group is an ethylgroup. Preferred substituted alkyl groups for R⁵ include an allyl group,a propargyl and a benzyl group.

R⁶, R⁷ and R⁸ preferably are independently (the same or different) aC₁₋₁₀ alkyl group, a C₂₋₁₀ alkenyl group, a C₂₋₁₀ alkynyl group, or anaryl group. A preferred substituted alkyl group for R⁶, R⁷ and R⁸ is a--(CH₂)_(N) R⁹ group, wherein N is an integer within the range of 1through 10 and R⁹ is a hydroxy group, an alkoxy group, an amino group, ahalogen atom, a cyano group or a nitro group. Preferred amino groups forR⁹ include alkylamino groups and a dialkylamino groups.

R¹¹ is preferably an alkylene group, an alkenylene or an alkynylenegroup. R¹² is preferably --CH═CH--CH₂ -- or --C.tbd.C--CH₂ --. X ispreferably Cl, Br or I. More preferably, X is Br or I. Most preferably,X is Br.

The present invention also provides a compound having the formula (2):##STR7## wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R¹¹ are as definedprior to this paragraph. The present invention further provides acompound of the above formula wherein one of R¹, R², R³, and R⁴ is notH. The present invention still further provides a compound of the aboveformula wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R¹¹ are as definedprior to this paragraph and wherein R⁵ is a methyl group, a C₃₋₁₀ alkylgroup, an allyl group, a benzyl group or a propargyl group.

The present invention further provides a compound having the followingformula (3): ##STR8##

The present invention further provides a compound having the followingformula (4): ##STR9##

The terms "alkyl", "aryl" and other groups refer generally to bothunsubstituted and substituted groups unless specified to the contrary.Unless otherwise specified, alkyl groups are hydrocarbon groups and arepreferably C₁ -C₁₅ (that is, having 1 to 15 carbon atoms) alkyl groups,and more preferably C₁ -C₁₀ alkyl groups, and can be branched orunbranched, acyclic or cyclic. The above definition of an alkyl groupand other definitions apply also when the group is a substituent onanother group (for example, an alkyl group as a substituent of analkylamino group or a dialkylamino group). The term "aryl" refers tophenyl or napthyl. As used herein, the terms "halogen" or "halo" referto fluoro, chloro, bromo and iodo.

The term "alkoxy" refers to --OR^(d), wherein R^(d) is an alkyl group.The term "aryloxy" refers to --OR^(e), wherein R^(e) is an aryl group.The term acyl refers to --C(O)R^(f). The term "alkenyl" refers to astraight or branched chain hydrocarbon group with at least one doublebond, preferably with 2-15 carbon atoms, and more preferably with 3-10carbon atoms (for example, --CH═CHR^(g)). The term "alkynyl" refers to astraight or branched chain hydrocarbon group with at least one triplebond, preferably with 2-15 carbon atoms, and more preferably with 3-10carbon atoms (for example, --C.tbd.CR^(h)). The terms "alkylene,""alkenylene" and "alkynylene" refer to bivalent forms of alkyl, alkenyland alkynyl groups, respectively.

The groups set forth above, can be substituted with a wide variety ofsubstituents to synthesize camptothecin analogs retaining activity. Forexample, alkyl groups may preferably be substituted with a group orgroups including, but not limited to, a benzyl group, a phenyl group, analkoxy group, a hydroxy group, an amino group (including, for example,free amino groups, alkylamino, dialkylamino groups and arylaminogroups), an alkenyl group, an alkynyl group and an acyloxy group. In thecase of amino groups (--NR^(a) R^(b)), R^(a) and R^(b) are preferablyindependently hydrogen, an acyl group, an alkyl group, or an aryl group.Acyl groups may preferably be substituted with (that is, R^(f) is) analkyl group, a haloalkyl group (for example, a perfluoroalkyl group), analkoxy group, an amino group and a hydroxy group. Alkynyl groups andalkenyl groups may preferably be substituted with (that is, R^(g) andR^(h) are preferably) a group or groups including, but not limited to,an alkyl group, an alkoxyalkyl group, an amino alkyl group and a benzylgroup.

The term "acyloxy" as used herein refers to the group --OC(O)R^(d).

The term "alkoxycarbonyloxy" as used herein refers to the group--OC(O)OR^(d).

The term "carbamoyloxy" as used herein refers to the group --OC(O)NR^(a)R^(b).

Amino and hydroxy groups may include protective groups as known in theart. Preferred protective groups for amino groups includetert-butyloxycarbonyl, formyl, acetyl, benzyl,p-methoxybenzyloxycarbonyl, trityl. Other suitable protecting groups asknown to those skilled in the art are disclosed in Greene, T., Wuts, P.G. M., Protective Groups in Organic Synthesis, Wiley (1991), thedisclosure of which is incorporated herein by reference.

In general, R¹, R², R³, R⁶, R⁷ and R⁸ are preferably not excessivelybulky to maintain activity of the resultant camptothecin analog.Preferably, therefore, R¹, R², R³, R⁶, R⁷ and R⁸ independently have amolecular weight less than approximately 250. More preferably R¹, R²,R³, R⁶, R⁷ and R⁸ independently have a molecular weight less thanapproximately 200.

Some of the camptothecin analogs of the present invention can beprepared for pharmaceutical use as salts with inorganic acids such as,but not limited to, hydrochloride, hydrobromide, sulfate, phosphate, andnitrate. The camptothecin analogs can also be prepared as salts withorganic acids such as, but not limited to, acetate, tartrate, fumarate,succinate, citrate, methanesulfonate, p-toluenesulfonate, and stearate.Other acids can be used as intermediates in the preparation of thecompounds of the present invention and their pharmaceutically acceptablesalts.

For purification, administration or other purposes, the E-ring (thelactone ring) may be opened with alkali metal such as, but not limitedto, sodium hydroxide or calcium hydroxide, to form opened E-ring analogsof compounds of formula (1) as set forth in the compounds of formula(4). The intermediates thus obtained are more soluble in water and maybe purified to produce, after treatment with an acid, a purified form ofthe camptothecin analogs of the present invention.

The E-ring may also be modified to produce analogs of compounds offormula (1) with different solubility profiles in water or othersolvents. Methods to achieve this goal include, but are not limited to,opening the E-ring with hydroxide or a water-soluble amino group orfunctionalizing the hydroxy group at position 20 of the E-ring with awater-soluble group such as a polyethylene glycol group. The analogsthus prepared act as pro-drugs. In other words, these analogs regeneratethe compounds of formula (1) (with the closed E-ring structure) whenadministered to a living organism. See, Greenwald, R. B. et al., J. Med.Chem., 39, 1938 (1996).

The analogs of the present invention are highly lipophilic and have beenshown to enhance activity both in vivo and in vitro. Moreover, theirA-ring substitution(s) have been shown to enhance blood stability.

The present invention also provides a method of treating a patient,which comprises administering a pharmaceutically effective amount of acompound of formulas (1) and/or (2) or a pharmaceutically acceptablesalt thereof. The compound may, for example, be administered to apatient afflicted with cancer and/or leukemia by any conventional routeof administration, including, but not limited to, intravenously,intramuscularly, orally, subcutaneously, intratumorally, intradermally,and parenterally. The pharmaceutically effective amount or dosage ispreferably between 0.01 to 60 mg of one of the compounds of formulas (1)and (2) per kg of body weight. More preferably, the pharmaceuticallyeffective amount or dosage is preferably between 0.1 to 40 mg of one ofthe compounds of formulas (1) and (2) per kg of body weight. In general,a pharmaceutically effective amount or dosage contains an amount of oneof the compounds of formulas (1) and (2) effective to displayantileukemic and/or antitumor (anticancer) behavior. Pharmaceuticalcompositions containing as an active ingredient of one of the compoundsof formulas (1) and (2) or a pharmaceutically acceptable salt thereof inassociation with a pharmaceutically acceptable carrier or diluent arealso within the scope of the present invention.

The present invention also provides a pharmaceutical compositioncomprising any of the compounds of formulas (1) and (2) and apharmaceutically acceptable carrier. The composition may, for example,contain between 0.1 mg and 3 g, and preferably between approximately 0.1mg and 500 mg of the compounds of formulas (1), (2) and/or (4), and maybe constituted into any form suitable for the mode of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a general synthetic scheme for thepreparation of compounds of formula (1).

FIG. 2 is an illustration of a synthesis of(20S)-11-fluoro-7-trimethylsilylcamptothecin.

FIG. 3 is an illustration of a synthesis of (20S)-10-acetoxy-7-trimethylsilylcamptothecin and (20S)-10-hydroxy-7-trimethylsilylcamptothecin.

FIG. 4 is an illustration of a synthesis of(20S)-10-amino-7-trimethylsilylcamptothecin.

FIG. 5 is an illustration of a synthesis of(20S)-10-amino-11-fluoro-7-trimethylsilylcamptothecin.

FIG. 6 is an illustration of a synthesis of a novel analog ofirinotecan.

FIG. 7 is an illustration of three representative silylcamptothecinanalogs of formula (2).

FIG. 8 is an illustration of the synthesis of a propargyl bromideprecursor.

FIG. 9 is an illustration of the synthesis of a radical precursor offormula (3).

FIG. 10 is an illustration of the reaction of the radical precursor ofFIG. 9 with three isonitriles.

FIG. 11 is an illustration of the final step of the synthesis of therepresentative silylcamptothecin analogs of FIG. 7.

FIG. 12 is an illustration of excitation and emission fluorescencespectra of 1 μM (20S)-7-[(2-trimethylsilyl)ethyl]camptothecin, 7-TMSEtCPT, (36c) (DB-172).

FIG. 13 is an illustration of emission fluorescence spectra of 1 μM(20S)-10-amino-7-[(2-trimethylsilyl)ethyl]camptothecin, 10-NH2-7-TMSEtCPT (DB-173).

FIG. 14 is an illustration of emission fluorescence spectra of 1 μM(20S)-10-hydroxy-7-[(2-trimethylsilyl)ethyl]camptothecin, 10-OH-7-TMSEtCPT (DB-174).

FIG. 15 is an illustration of fluorescence spectra of 1 μM(20S)-10-hydroxy-7(tert-butyldimethylsilyl)camptothecin, 10-OH-7-TBS CPT(DB-67) in ethanol, 0.29 M DMPG and PBS.

FIG. 16 is an illustration of equilibrium binding of camptothecinanalogs to DMPC.

FIG. 17 is an illustration of equilibrium binding of highly lipophiliccamptothecin analogs of the present invention to DMPC.

FIG. 18 is an illustration of equilibrium binding of highly lipophiliccamptothecin analogs of the present invention to DMPG.

FIG. 19 is an illustration of double-reciprocal plots for the binding ofhighly lipophillic camptothecin analogs of the present invention to DMPCsmall unilamellar vesicles (SUVs) at 37° C.

FIG. 20 is an illustration of double-reciprocal plots for the binding ofhighly lipophillic camptothecin analogs of the present invention to DMPGSUVs at 37° C.

FIG. 21 is an illustration of the stability of DB-172 in PBS buffer pH7.4 at 37° C.

FIG. 22 is an illustration of the dependence of fluorescence intensityof DB-172 on time and drug concentration.

FIG. 23 is an illustration of the fluorescence intensity of DB-172 independence on time and concentration.

FIG. 24 is an illustration of the dependence of the total fluorescenceintensity on time and pH for the carboxylate form of DB-172.

FIG. 25 is an illustration of the dependence of total fluorescenceintensity on time for several camptothecin analogs of the presentinvention.

FIG. 26 is an illustration of the drug stability of several camptothecinanalogs of the present invention in phosphate buffered saline (PBS) andhuman blood.

FIG. 27 is an illustration of the drug stability of several camptothecinanalogs of the present invention in PBS, whole blood and PBS/human serumalbumin (HSA).

FIG. 28 is an illustration of the plasma concentration of DB-67 afteroral dosage.

DETAILED DESCRIPTION OF THE INVENTION

Compounds

Among the compounds of formulas (1) and (2), those having the(S)-configuration at position 20 of the E-ring are preferred forpharmaceutical use.

R¹ and R² are preferably and independently (the same or different) H, ahydroxy group, a halo group, an amino group, a nitro group, a cyanogroup, a C₁₋₃ alkyl group, a C₁₋₃ perhaloalkyl group, a C₁₋₃ alkenylgroup, a C₁₋₃ alkynyl group, a C₁₋₃ alkoxy group, a C₁₋₃ aminoalkylgroup, a C₁₋₃ alkylamino group, a C₁₋₃ dialkylamino group, or R¹ and R²together form a group of the formula --O(CH₂)_(n) O-- wherein nrepresents the integer 1 or 2. More preferably, R¹ and R² areindependently (the same or different) H, a methyl group, an amino group,a nitro group, a cyano group, a hydroxy group, a hydroxymethyl group, amethylamino group, a dimethylamino group, an ethylamino group, adiethylamino group, an aminomethyl group, a methylaminomethyl group, adimethylaminomethyl group, and the like.

R³ is preferably F, an amino group, or a hydroxy group. R⁴ is preferablyH, a trialkylsilyl group or F. R⁵ is preferably an ethyl group. R⁶, R⁷and R⁸ are preferably independently (the same or different) a C₁₋₆ alkylgroup, a phenyl group or a --(CH₂)_(N) R¹⁰ group, wherein N is aninteger within the range of 1 through 6 and R¹⁰ is a halogen or a cyanogroup.

Method of Preparation

The compounds of formula (1) of the present invention can be preparedaccording to the general synthetic scheme shown in FIG. 1. In thesynthetic scheme of FIG. 1, an iodopyridone (2) is first N-alkylatedwith a propargyl derivative (3) to produce radical precursor (4).Radical precursor (4) then undergoes a radical cascade witharylisonitrile (5) to generate product (1). The N-alkylation proceedssmoothly following optimized conditions. See Curran, D. P. et al.,Tetrahedron Lett., 36, 8917 (1995), the disclosure of which isincorporated herein by reference. The synthesis of iodopyridone (2) andthe conditions of the radical cascade have been previously reported. Thepropargylating agent (3) is readily prepared by the standard silylationof the dianion of propargyl alcohol with a suitable silylating agent R⁶R⁷ R⁸ SiX followed by conversion of the propargyl alcohol to a leavinggroup such as a bromide, iodide or sulfonate. See Curran, D. P. et al.,Angew. Chem. Int. Ed. Engl., 34, 2683 (1995), the disclosure of which isincorporated herein by reference, and U.S. patent application Ser. No.08,436,799, filed May 8, 1995, the disclosures of which are incorporatedherein by reference.

Generally, various reagents can be used in the radical cascadeincluding, but not limited to, hexamethylditin, hexamethyldisilane, ortetrakis(trimethylsilyl)silane. The source of energy for this reactioncan be a sun lamp or an ultraviolet lamp. The temperature is preferablyset between approximately 25 and 150° C. More preferably, thetemperature is set at approximately 70° C. There are generally nolimitations upon the choice of solvent used other than inertness to theradical cascade. Preferred solvents include benzene, toluene,acetonitrile, THF and tert-butanol. Also, there is very broad latitudein the choice of substituents on the alkyne and the isonitrile becauseof the mildness of the reaction conditions.

FIG. 2 illustrates an embodiment of a general synthetic scheme for thesynthesis of (20S)-11-fluoro-7-trimethylsilylcamptothecin (12). Aproblem in this synthetic scheme is to control the regioselectivity ofthe radical cascade when both ortho positions in the arylisonitrile areavailable for cyclization (that is, R⁴ is H in the final compound offormula (1)). One solution to this problem relies upon the introductionof a trimethylsilyl group on the aryl isonitrile, (e.g.3-fluoro-2-trimethylsilylphenyl isonitrile (9)). The trimethylsilylsubstituent blocks one of the ortho sites of the isonitrile towardcyclization and can be removed after the cascade reaction byhydrodesilylation. In this example, the selectivity proceeds further inthe sense that only one of the trimethylsilyl groups is removed in thelast step.

Other embodiments of the general synthetic scheme for the preparation ofseveral novel camptothecin derivatives of formula (1) are illustrated inFIGS. 3 to 6, and in the Examples.

The preparation of the compounds of formula (2) is illustrated in FIGS.7 through 11. In that regard, three representative, novel A,B ringsubstituted silylcamptothecin compounds (36a), (36b), and (36c) areillustrated in FIG. 7.

The first step in the synthesis of these analogs was to preparepropargyl bromide (41) as illustrated in FIG. 8. Swern oxidation of thecommercially available trimethylsilylpropanol (37) gavetrimethylsilylpropanal (38) in 85% yield. Sakar, T. K. et al.,Tetrahedron 46, 1885 (1990). Following procedure A of Corey, E. J. andFuchs, P. L., Tetrahedron Lett., 36, 3769 (1972), aldehyde (38) wasconverted to the dibromoolefin (39) in 55% yield. Piers, E. and Gaval,A. V., J. Org. Chem., 55, 2374 (1990). Addition of 2 equivalents ofn-BuLi at -78° C. in THF followed by warming to 22° C. and the quenchingwith paraformaldehyde at reflux to give (40) in 84% yield. Finally, asolution of triphenylphosphine and Br₂ in anhydrous CH₂ Cl₂ gave thepropargyl bromide (41) in 87% yield.

With the preparation of propargyl bromide (41) completed, radicalprecursor (43) was prepared as illustrated in FIG. 9. Following anN-alkylation procedure, (42) was alkylated with (41) to give the desiredradical precursor (43) in 74% yield. Reaction of (43) with therespective isonitrile (44a) or (44b) gave the protectedsilylcamptothecin derivatives (45a) and (45b) in 55% and 56% yields,respectively, as illustrated in FIG. 10. Finally, deprotection of (45a)with 2 equivalents of K₂ CO₃ in MeOH/H₂ O solution gave a 47% yield ofthe 10-hydroxy derivative (36a) as illustrated in FIG. 10. Finally,treatment with trifluoroacetic acid in CH₂ Cl₂ converted (45b) to the10-amino derivative (36b) in 65% yield.

The method of the present invention also provides ready synthesis of(20S)-7-[(2-trimethylsilyl)ethyl]camptothecin (36c, FIG. 7). Reaction ofphenyl isonitrile with iodo pyridone (43) gives this derivative in 52%yield (FIG. 10). The (20S)-7-[(2-trimethylsilyl)ethyl]camptothecin (36c)and (20S)-7-(2-trimethylsilyl)camptothecin (disclosed in U.S. patentapplication Ser. No. 08/436,799) structures have recently been describedby Hausheer et al. in International Patent Application PublicationNumber WO 98/07727. It appears that the characterization informationregarding these compounds set forth in International Patent ApplicationPublication Number WO 98/07727 is not correct. Specifically, thespectroscopic data provided for both of these compounds are inconsistentwith the assigned structures and do not match in any respect thespectroscopic data reported in the examples of this patent.Additionally, the spectroscopic data for all of the silyl-containingcamptothecins in WO 98/07727 appear to be inconsistent with the assignedstructures.

The present invention thus provides a short and efficient syntheticscheme well suited to known structure-activity relationships in thecamptothecin family. Indeed, the biological activity of the camptothecinskeleton is generally intolerant or has very little tolerance tosubstituents other than at the 7 and/or 9-11 positions. Followingsynthesis, these substituents are introduced via the alkynylderivative(3) and arylisonitrile (5), respectively.

Antitumor Activities and Human Blood Stability Characteristics

The antitumor activities of several compounds of formula (1) are shownin Table 1 and compared to those of several well known camptothecinanalogs using several known assays as described further below. Thesyntheses of the various exemplary compounds of the present inventionset forth in Table 1 are discussed in further detail in an Examplesection following this section.

                                      TABLE 1                                     __________________________________________________________________________    Biological Activities of (20S)-7-Silyl-Camptothecin Derivatives                                                                    Inhibition of cancer                                                        cell growth  IC.sub.50                                                        (nm) Enhancement of                                                           Topo I Inhibition of                                                          Topo I                     Example                                                                            7.sup.a                                                                            9  10  11  12  HL-60                                                                             833K  DC-3F                                                                             Mediated DNA Cleavage                                                                     mediated DNA relaxation    __________________________________________________________________________    CPT  H    H  H   H   H   5   10    6-9 +++         +++                          IRT Et H OPP.sup.a H H 270 487 372 - -                                         1 TMS H H H H 3.8 5.6 4.2 ++++ +++                                            2 TBDMS H H H H 0.12 1.2 2.9 ++++ +++                                         3 TBDPS H H H H 339 243 663 ++ +                                              4 TMS H OAc H H 2.7  6.7 ++++ +++++                                           5 TMS H OH H H 2.6 7.0 6.9 ++++ +++++                                       5a  Example 5 with opened E ring                                                                      9.7 15.0  14.2                                                                              +++         +                           6   TMS  H  OPP.sup.a                                                                         H   H   66  214   256 -           -                             7 TMS H H F H 0.75 0.92 2.0 ++++ +++++                                        7a TMS F H H H 3.0 2.9 8.2 ++++ ++++                                          TMS H H F H                                                                       (2:1)                                                                     8 TMS H NH.sub.2 H H 0.52 5.7 0.72 - -                                        9 TMS H H NH.sub.2 H 2.6 7.4 6.4 - -                                         10 TMS H NH.sub.2 F H 0.07 0.14 0.29 ++++ ++++                                11 TMS H H F F 1.01 2.1 2.5 +++ +++                                            TMS F F H H                                                                       (3/1)                                                                    12 TIPS H H H H 1506 10730 1038 - -                                           13 TES H H H H 31.9 122 57.1 - -                                              14 DMNPS H H H H 66.9 197 64.1 - -                                            15 DMCPS H H H H 0.91 2.7 2.7 - -                                             16 DMHPS H H H H 2.1 5.4 2.3 - -                                              17 TBDMS H OAc H H 1.86 -- 3.57 - -                                           18 TBDMS H OH H H 2.60 -- 5.20 - -                                          __________________________________________________________________________     .sup.a OPP = irinotecan's pyrrolidinyl pyrrolidine carbamate;                 TMS = trimethylsilyl;                                                         TBDMS = tbutyldimethylsilyl;                                                  TBDPS = tbutyldiphenyl silyl;                                                 TES = triethylsilyl;                                                          TIPS = triisopropylsilyl;                                                     DMNPS = dimethylynorpinylsilyl;                                               DMCPS = dimethyl3-cyanopropylsilyl;                                           DMHPS = dimethyl3-halopropylsilyl;                                            .sup.b More active than CPT in S180 in BD.sub.2 F.sub.1 mice testing.         .sup.c More active than CPT in Lewis lung Carcinoma in BD.sub.2 F1 mice.      The designation "--" means "not determined."-                            

As illustrated in Table 1, the compounds of the present inventionexhibit good to excellent antitumor activity as compared to camptothecin(CPT) and irinotecan (IRT).

Cytotoxicity Assays

The camptothecin derivatives were evaluated for their cytotoxic effectson the growth of HL-60 (human promyelocytic leukemic), 833K (humanteratocarcinoma) and DC-3F (hamster lung) cells in vitro. The cells werecultured in an initial density of 5×10⁻⁴ cell/ml. They were maintainedin a 5% CO₂ humidified atmosphere at 37° C. in RPMI-1640 media(GIBCO-BRL Grand Island, N.Y.) containing penicillin100u/ml)/streptomycin (100 μg/ml) (GIBCO-BRL) and 10% heat inactivatedfetal bovine serum. The assay was performed in duplicate in 96-wellmicroplates. The cytotoxicity of the compounds toward HL-60 cellsfollowing 72 hr incubation was determined by XTT-microculturetetrazolium assay. Scudiero, D. A., et al., Cancer Res., 48, 4827(1988), the disclosure of which is incorporated herein by reference.2',3'-bis(-methoxy-4-nitro-5-sulfheny)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT) was prepared at 1 mg/ml in prewarmed (37° C.) mediumwithout serum. Phenazine methosulfate (PMS) and fresh XTT were mixedtogether to obtain 0.075 mM PMS-XTT solution (25 μl of the stock 5 mMPMS was added per 5 ml of 1 mg/ml XTT). Fifty μl of this mixture wasadded to each well of the cell culture at the end of 72 hr incubation.After incubation at 37° C. for 4 hr., absorbance at 450 nm and 630 nmwas measured with a microplate reader (EL340, Bio-Tek Instruments, Inc.,Winooski, Vt.).

The cytotoxicity of the camptothecin compounds toward 833Kteratocarcinoma solid tumor cells and DC-3F hamster lung cells wasdetermined in 96-well microplates by a method described by Skehan et al.for measuring cellular protein content. Skehan et al., "New ColorometricCytotoxicity Assay for Anticancer Drug Screening," J. Nat'l CancerInst., 82, 1107 (1990), the disclosure of which is incorporated hereinby reference. Cultures were fixed with trichloroacetic acid and thenstained for 30 minutes with 0.4% sulforhodamine B dissolved in 1% aceticacid. Unbound dye was removed by acetic acid washes, and theprotein-bound dye was extracted with an unbuffered Tris base[tris(hydroxy-methyl)aminomethan] for determination of absorbance at 570nm in a 96-well microplate reader. The experiments were carried out induplicate using five to six concentrations of the drugs tested. Datawere analyzed via computer software. See, Chou, J, and Chou, T. C.,Dose-Effect Analysis With Microcomputers: Quantitation of ED₅₀, LD₅₀,Synergism, Antagonism, Low-Dose Risk, Receptor-Ligand Binding and EnzymeKinetics, 2^(nd) ed., Biosoft, Cambridge (1987); and Chou, T. C., "TheMedian-Effect Principle and the Combination Index for Quantitation ofSynergism and Antagonism," Synergism and Antagonism in Chemotherapy,Academic Press, San Diego, 61-102 (1991), the disclosures of which areincorporated herein by reference.

Topo I Mediated DNA Cleavage Assay

For DNA cleavage assay the reaction mixture comprised Tris-HCl buffer 10mM, pH7.5; PBR₃₂₂ supercoiled double stranded circular DNA (4363 basepairs, from Bochringer Mannheim Biochemicals) 0.125 μg/ml, drug(camptothecin or its derivatives) concentration at 1, 10 and 100 μM, inthe presence of purified DNA topoisomerase I with final volume of 20 μlas described previously. Hsiang, Y. H., et al., "Camptothecin InducesProtein-Linked DNA Breaks Via Mammalian DNA Topoisomerase I," J. Biol.Chem., 260, 14873 (1985), the disclosure of which is incorporated hereinby reference. Incubation was carried out at 37° C. for 60 min. Thereaction was stopped by adding the loading buffer dye (2% sodium dodesylsulfate, 0.05% bromophenol blue and 6% glycerol). Electrophoresis wascarried cut on 1% agarose gel plus ethidium bromide (1 μg/ml) in TBEbuffer (Tris-base-boric acid-EDTA) and ran at 25 V for 18 hrs.Photographs were taken under UV light using Polaroid film type 55/N anddeveloped as indicated by the manufacturer.

Inhibition of Topo I Mediated Relaxation of Supercoiled DNA

To study the inhibiting effect on DNA topoisomerase I mediatedrelaxatioon of DNA, the method described by Liu and Miller was used.Liu, H. F. et al., "Cleavage of DNA by Mammalian DNA Topoisomerase II,"J. Biol. Chem., 258, 15365 (1980), the disclosure of which isincorporated herein by reference. For this assay, 0.18 μg of PBR₃₂₂ DNA,0.5 U of Topo I (GIBCO-BRL), various concentrations (1-100 μM ofcamptothecin or an analog, in a reaction mixture (201l) containing 50 mMTris-HCl, pH 7.5, 120 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.5 mM EDTA, 30μg/ml BSA, 20 μg/ml PBR₃₂₂ DNA and various amounts of the enzyme wasincubated at 37° C. for 30 min., and stopped with 5% SBS and 150 μg/mlproteinase K. The samples were loaded onto 1% agarose in TAE runningbuffer, electrophoresed overnight at 39 V, stained with EtBr, andphotographed under UV light.

Antitumor Activity In Vivo

Antitumor activities of camptothecin derivatives were tested in B₆ D₂ F₁mice bearing sarcoma-180 or Lewis lung murine solid tumor. For S-180,3×10⁶ cells were innoculated subcutaneously on day 3. Antitumortreatment started on day 1 intraperitoneously twice daily for five days.Tumor volumes on day 7 and day 14 were measured. Average tumor volumeswere described as the ratio of treated versus untreated control (T/C).The control (treated with DMSO vehicle only) tumor volumes for day 7 andday 14 were 0.11 cm³ and 0.61 cm³, respectively. The T/C camptothecin isdesignated with "+++." An increment or decrement of 10% as compared tothe camptothecin T/C on day 14 at 2 mg/kg dosage is designated withincrease or decrease of one "+" unit, respectively.

For Lewis lung carcinoma, tumor cells (1×10⁶) were inoculatedsubcutaneously on day 0 and treatment started on day 1,intraperitoneously twice daily for five days. The grading of effects wasas described above.

As shown Table 1, many of the camptothecin derivatives of formula (1)tested for the antitumor cytotoxicity in vitro exhibited higher potencythan camptothecin in one to three cell lines. Most of those compoundsexhibiting higher antitumor cytotoxicity also exhibited higher potencyin enhancing the DNA-topoisomerase I-mediated cleavage of PBR₃₂₂ DNA, orin inhibiting the DNA-topoisomerase I-mediated relaxation of PBR₃₂₂ DNA.These results suggest excellent correlation between the antitumorcytoxicity of the camptothecin compounds with their ability to inhibitthe functions of DNA-topoisomerase I.

For in vivo chemotherapeutic effects in tumor-bearing mice, for example,7-trimethylsilyl camptothecin showed better activity than camptothecinagainst sarcoma 180 in B₆ D₂ F₁ mice at several equivalent doses in adose dependent manner in terms of tumor volume reduction. Similarly, forLewis lung carcinoma, 7-trimethylsilyl-11-flouro camptothecin exhibiteda similar antitumor effect to camptothecin in terms of tumor volumereduction at 4-fold lower doses than camptothecin. Thus,7-trimethylsilyl-11-flouro camptothecin is more efficacious thancamptothecin in its antitumor effects in vivo.

Stability in Human Blood

Recently the intrinsic fluorescent emissions from the lactone andcarboxylate forms of camptothecin have been studied to elucidate theirmarkedly different interactions with human blood components. Burke, T.G. and Mi, Z., "Ethyl substitution at the 7 position extends thehalf-life of 10-hydroxycamptothecin in the presence of human serumalbumin," J. Med. Chem. 36: 2580-2582 (1993); Burke, T. G., Mishra, A.K., Wani, M. C. and Wall, M. E., "Lipid bilayer partitioning andstability of camptothecin drugs," Biochemistry. 32: 5352-5364 (1993);Burke, T. G. and Mi, Z.: "Preferential Binding of the Carboxylate Formof Camptothecin by Human Serum Albumin," (1993) Anal. Biochem. 212,285-287; Burke, T. G. and Mi, Z., "The Structural Basis of CamptothecinInteractions with Human Serum Albumin: Impact on Drug Stability," (1994)J. Med. Chem. 37, 40-46; Burke, T. G. Munshi, C. B., Mi, Z., and Jiang,Y., "The Important Role of Albumin in Determining the Relative HumanBlood Stabilities of the Camptothecin Anticancer Drugs," (1995) J.Pharma. Sci. 84, 518-519; Mi, Z. and Burke, T. G., "DifferentialInteractions of Camptothecin Lactone and Carboxylate Forms with HumanBlood Components," (1994) Biochemistry, 33, 10325-10336; Mi, Z. andBurke, T. G., "Marked Interspecies Variations Concerning theInteractions of Camptothecin with Serum Albumins: A Frequency-DomainFluorescence Spectroscopic Study," (1994) Biochemistry 33, 12540-12545;Mi, Z., Malak, H., and Burke, T. G., "Reduced Albumin Binding Promotesthe Stability and Activity of Topotecan in Human Blood," (1995)Biochemistry, 34, 13722-13728, the disclosures of which are incorporatedherein by reference.

In phosphate buffered saline (PBS) at pH 7.4, frequency-domainfluorescence lifetime spectroscopy reveals that human serum albumin(HSA) preferentially binds the carboxylate form of camptothecin with a200-fold higher affinity than the lactone form. These interactionsresult in camptothecin opening more rapidly and completely in thepresence of HSA than in the absence of the protein. In human plasma, pH7.4 and 37° C., camptothecin lactone opens rapidly and completely to thecarboxylate form with a t_(1/2) value of 11 min and an almost negligible% lactone at equilibrium value of 0.2%. In whole blood versus plasma,camptothecin displayed enhanced stability (t_(1/2) value of 22 min and a% lactone at equilibrium value of 5.3%). The enhanced stability ofcamptothecin lactone in human blood was found to be due to drugassociations with the lipid bilayers of red blood cells. Camptothecinbinds erythrocyte membranes, the drug localizes within the acyl chainregion, and accordingly remains protected from hydrolysis.

The human blood stabilities of the several camptothecin analogues ofclinical interest have been compared. As was observed in the case ofcamptothecin, 9-aminocamptothecin was observed to hydrolyze almostcompletely (>97%) in PBS solution containing HSA. Although no attemptwas made to spectroscopically quantify the relative binding affinitiesof the lactone and carboxylate forms of the 9-amino congener due totheir significantly reduced fluorescence quantum yields relative tocamptothecin, HPLC data were consistent with HSA preferentially bindingthe carboxylate form of this agent over its lactone form. In plasma itwas observed that >99.5% of the 9-amino analog converted to carboxylate,a finding which again closely parallels stability data obtained usingcamptothecin. In whole blood, <0.5% and 5.3% are the fractions of9-aminocamptothecin and camptothecin, respectively, which remained inthe lactone form at equilibrium. The approximately 10-fold higher levelof lactone remaining at equilibrium for camptothecin relative to9-aminocamptothecin may, in part, be accounted for by the enhancedlipophilicity and greater ability of camptothecin to transition from theaqueous environment and into erythrocyte membranes present in wholeblood.

In stark contrast to the low levels of lactone remaining at equilibriumin whole human blood for camptothecin and 9-aminocamptothecin (<0.5% and5.3%, respectively), topotecan (11.9%), CPT-11 (21.0%), and SN-38(19.5%) all display improved blood stabilities. While lactone levels atequilibrium for topotecan are 20-fold greater than for9-aminocamptothecin, the corresponding levels of lactone for IRT(CPT-11) and 10-hydroxy-7-ethylcamptothecin (SN-38) are approximately40-fold greater than in the case of 9-aminocamptothecin. The significantgains in the relative stabilities of topotecan, CPT-11, and SN-38 can becorrelated to their favorable interactions with HSA. These agentscontain structural substituents at the 7- and 9- positions which hinderand prevent the preferential binding of the carboxylate drug forms byHSA. The technique of time-resolved fluorescence anisotropy has recentlybeen used to demonstrate that, under experimental conditions wherecamptothecin carboxylate associates with HSA and tumbles in solutionclosely associated with the protein, the carboxylate forms of topotecanand CPT-11 do not associate with HAS. In the case of SN-38, directspectroscopic evidence has been obtained which indicates that HSApreferentially binds the lactone form of this agent, thereby shiftingthe lactone-carboxylate equilibrium to the lactone.

Thus, it is clear from these observations that HSA plays an importantrole in determining the relative human blood stabilities of thecamptothecins. In the cases of camptothecin and 9-aminocamptothecin, theprotein acts as a sink for the carboxylate drug form, binding the openedring species and thereby shifting the lactone-carboxylate equilibria tothe carboxylate. However, in the cases of topotecan, CPT-11, and SN-38,no such preferential binding of the carboxylate drug form by HSA isobserved. Opposite to the situation with camptothecin and its 9-aminoanalogue, HSA preferentially binds the lactone form of SN-38 whichthereby promotes higher circulatory levels of this biologically activespecies.

The rapid and extensive loss of active drug that occurs with currentlyclinically relevant camptothecins indicates that it would be highlyadvantageous to identify camptothecins with reduced protein bindinginteractions and improved human blood stabilities. In that regard, thecamptothecin analogs of the present invention exhibit unique propertiesthat result in the agents displaying improved human blood stabilitieswhile maintaining high anticancer activities.

Experimental Methods for the Determination of Lipid Bilayer Partitioning(i.e. Lipophilicity) and Lactone Ring Stability.

Chemicals. All camptothecin analogs were in the 20(S) configuration andwere of high purity (>98%) as determined by HPLC assays withfluorescence detection. All other agents were reagent grade and wereused without further purification. High purity water provided by aMilli-Q UV PLUS purification system (Bedford, Mass.) was utilized in allexperiments.

Drug Stock Solution Preparation. Stock solutions of the drugs wereprepared in dimethylsulfoxide (A.C.S. spectrophotometric grade, Aldrich,Milwaukee, Wis.) at a concentration of 2×10⁻³ M and stored in dark at 4°C. L-α-Dimyristoylphosphatidylcholine (DMPC) andL-α-dimyristoylphosphatidylglycerol (DMPG) were obtained from AvantiPolar Lipids, Alabaster, Ala., and were used without furtherpurification. All other chemicals were reagent grade and were usedwithout further purification.

Vesicle Preparation. Small unilamellar vesicle (SUV) suspensions wereprepared the day of an experiment by the method of Burke and Tritton,"The Structure Basis of Anthracycline Selectivity for UnilamellarPhophatidylcholine Vesicles: An Equilibrium Binoinl Study," Biochem24:1768-1776 (1985). Briefly, stock lipid suspensions containing 200mg/mL lipid in phosphate buffered saline (PBS, pH 7.4) were prepared byVortex mixing for 5-10 min above the TM of the lipid. The lipiddispersions were then sonicated using a bath-type sonicator (LaboratorySupplies Co., Hicksville, N.Y.) for 3-4 h until they became opticallyclear. A decrease in pH from 7.4 to 6.8 was observed for the SUVpreparations of DMPG; therefore, the pH of these SUV suspensions wasadjusted to 7.4 using small quantities of 2.5 M NaOH in PBS, followed byadditional sonication. Each type of vesicle suspension was annealed for30 min at 37° C. and then used in an experiment.

Fluorescence Instrumentation. Steady-state fluorescence measurementswere obtained on a SLM Model 9850 spectrofluorometer with a thermostatedcuvette compartment. This instrument was interfaced with an IBM PS/2model 55 SX computer. Excitation and emission spectra were recorded withan excitation resolution of 8 nm and an emission resolution of 4 nm. Inall cases spectra were corrected for background fluorescence and scatterfrom unlabeled lipids or from solvents by subtraction of the spectrum ofa blank. Steady-state fluorescence intensity measurements were made inthe absence of polarizers. Steady-state anisotropy (a) measurements weredetermined with the instrument in the "T-format" for simultaneousmeasurement of two polarized intensities. The alignment of polarizerswas checked routinely using a dilute suspension of 0.25 μm polystyrenemicrospheres (Polysciences, Inc, Warrington, Pa.) in water andanisotropy values of >0.99 were obtained. Alternatively, polarizerorientation was checked using a dilute solution of glycogen in water.The anisotropy was calculated from a=(I_(VV) -GI_(VH))/(I_(VV)+GI_(VH)), where G=I_(VH) /I_(HH) and the subscripts refer to verticaland horizontal orientations of the excitation and emission polarizers,respectively.

Anisotropy measurements for camptothecins were conducted using excitinglight of 370 nm and long pass filters on each emission channel in orderto isolate the drug's fluorescence signal from background scatter and/orresidual fluorescence. All emission filters were obtained from OrielCorp (Stamford, Conn.). The combination of exciting light and emissionfilters allowed adequate separation of fluorescence from backgroundsignal. The contribution of background fluorescence, together withscattered light, was typically less than 1% of the total intensity.Since the lactone rings of camptothecin and related congeners undergohydrolysis in aqueous medium with half-lives of approximately 20 min.,all measurements were completed within the shortest possible time (ca.0.5 to 1 min) after mixing the drug stock solution with thermallypre-equilibrated solutions such that the experiments were free ofhydrolysis product.

Determination of Equilibrium Binding Constants. The method offluorescence anisotropy titration reported in Burke, T. G., Mishra, A.K., Wani, M. C. and Wall, M. E. "Lipid bilayer partitioning andstability of camptothecin drugs," Biochemistry. 32: 5352-5364 (1993) wasemployed to determine the concentrations of free and bound species ofdrug in liposome suspensions containing a total drug concentration of1×10⁻⁶ M and varying lipid concentrations. All experiments wereconducted in glass tubes. The overall association constants are definedas K=[A_(B) ]/[A_(F) ][L] where [A_(B) ] represents the concentration ofbound drug, [A_(F) ] represents the concentration of free drug, and [L]represents the total lipid concentration of the sample.Double-reciprocal plots of the binding isotherms {1/(bound fraction ofdrug) vs. 1/[lipid]} were linear and K values were determined from theirslopes by the method of linear least squares analysis. A computerprogram based on the K=[A_(B) ]/[A_(F) ][L] relationship was written topredict bound drug levels for specified values of K and total drug.

Kinetics of Lactone Ring Opening. The hydrolysis kinetics ofcamptothecins in the presence of different blood components weredetermined by a quantitative C18 reversed-phase high-performance liquidchromatography (HPLC) assay as described in the literature. Mi and Burke(1994), supra. The preparation of whole blood and fractionated bloodsamples was carried out as described previously. Crystallized HSA ofhigh purity (>97%) from Sigma Chemical (St. Louis, Mo.) was used. HSAstock solutions were prepared in PBS buffer with a final pH of7.40±0.05. HSA concentrations were determined by UV absorbance at 278 nmusing an extinction coefficient of 39,800 M⁻¹ cm⁻¹ (Porter, 1992). Allother agents were reagent grade and were used without furtherpurification. High purity water provided by a Milli-Q UV PLUSpurification system (Bedford, Mass.) was utilized in all experiments.

Anticancer Activities of Highly Lipophilic Camptothecins of the PresentInvention Determined In Vitro in Cell Culture Experiments

Cells. Cytotoxicity measurements were conducted using MDA-MB-435tumorigenic human breast cancer cells. The cells were exposed to a rangeof drug concentrations for 72 hr exposure periods and then viability wasassessed using a sulphorrhodamine B (SRB) assay. SRB assays wereperformed using a standard assay.

Fluorescence Anisotropy Titration Demonstrates that the CamptothecinAnalogs of the Present Invention Display Exceptionally High EquilibriumAssociation Constants for Lipid Vesicles

FIGS. 12 through 15 depict the fluorescence excitation and emissionspectra of several of the new camptothecin analogs. FIG. 12 summarizesthe excitation and emission spectra of 1 μM DB-172 in phosphate bufferedsaline solution. The figure indicates that upon introduction of lipidbilayers into the sample there is an increase in the fluorescenceemission of the compound, indicative of an interaction between the drugand the membrane. Upon changing the solvent to ethanol the fluorescencealso changes. FIGS. 13 through 15 summarize the emission spectra ofDB-173, DB-174, and DB-67, respectively, in the presence and absence ofmembranes. In each case there is a marked increase in fluorescenceintensity as the drug partitions into the lipid bilayer. In each casethere is also a prominent blue-shifting or shift in the emission spectrato lower wavelength upon drug interaction with membrane. The spectraldata presented in FIGS. 12 through 15 clearly indicate that the newagents are fluorescent and the spectral parameters of the drugs changeupon addition of lipid bilayer membranes to the samples. Table 2compares the maximum excitation and emission wavelengths of newcamptothecin analogs with congeners that have been made previously. Theintrinsic fluorescent nature of the camptothecins allows for thesensitive method of steady-state fluorescence anisotropy titration to beemployed to determine the strength of the binding interactions of thevarious analogs with lipid bilayers.

                  TABLE 2                                                         ______________________________________                                        Fluorescence Spectral Parameters for camptothecin analogs                       in solution and bound to DMPC and DMPG SUVs                                                  Excitation                                                                             Emission                                              (nm) (nm)                                                                   Compound (S)     PBS      PBS    DMPC  DMPG                                   ______________________________________                                        camptothecin     367      430    422   415                                      7-methylcamptothecin 366 421 418 405                                          7-ethylcamptothecin 367 422 419 406                                           7-propylcamptothecin 366 422 419 406                                          7-methyl-10-ethoxycamptothecin 376 430 401 400                                7-ethyl-10-methoxycamptothecin 376 430 403 406                                7-propyl-10-methoxycamptothecin 376 430 404 404                               DB-67 400 550 445 440                                                         DB-172 370 424 424 422                                                        DB-173 395 526 503 502                                                        DB-174 380 550 433 431                                                        DB-202 377 450 439 437                                                        CHJ-792 400 531 517 519                                                     ______________________________________                                    

The following designations are used herein: DB-172,(20S)-7-[(2-trimethylsilyl)ethyl]camptothecin (36c); DB-173,(20S)-10-amino-7-[(2-trimethylsilyl)ethyl]camptothecin (36b); DB-174,(20S)-10-hydroxy-7-[(2-trimethylsilyl)ethyl]camptothecin (36a); DB-67,(20S)-10-hydroxy-7(tert-butyldimethylsilyl)camptothecin; DB-148,(20S)-7-(3-chloropropyldimethylsilyl)camptothecin; DB-158,(20S)-10-hydroxy-7-(3-chloropropyldimethylsilyl) camptothecin; DB-202,(20S)-7(tert-butyldimethylsilyl) camptothecin; CHJ-792,10-amino-7-trimethylsilylcamptothecin (20); DB-124,10-hydroxy-7-(3-dimethylaminopropyldimethylsilyl) camptothecinhydrochloride salt; and DB-104, 7-(3-dimethylaminopropyldimethylsilyl)camptothecin hydrochloride salt.

A steady-state fluorescence anisotropy (a) measurement is related to therotational rate of the fluorescent molecule through the Perrin Equation:

    a.sub.o /a=1+(τ/Φ)

where a_(o) is the limiting fluorescence anisotropy in the absence ofdepolarizing rotations, τ is the excited-state lifetime, and Φ is therotational correlation time of the fluorophore. The above equationstates that changes in either the τ or Φ values of a fluorescentcompound can modulate its steady-state anisotropy.

The excited-state lifetime values of camptothecin in PBS, glycerol, andmethanol were examined at 37° C. The lifetime values were determined tobe 4.7 ns, 3.8 ns, and 3.5 ns, respectively. Similarly, camptothecin'slifetime value when associated with DMPC bilayers were measured at 37°C., and the average value for membrane-bound drug was found to be 3.7ns.

Thus the lifetime measurements described above indicate thatcamptothecin's excited-state lifetime is relatively insensitive toalterations in microenvironment (e.g. a change in solvent or fluorophorerelocation from an aqueous milieu to a phospholipid membrane). For afluorophore whose τ value remains relatively constant during atransition which strongly impacts on its rotational motion (such as achange in solvent viscosity or fluorophore binding to largemacromolecular assemblies such as liposomal particles), the Perrinequation indicates a direct relationship between a and Φ values willexist (that is, as the Φ value of the fluorescent compound increases,then so too does its steady-state anisotropy value).

Steady-state fluorescence anisotropy values of the camptothecinanalogues are highly sensitive to solvent viscosity and to associationswith small unilamellar lipid vesicles. For example, topotecan has an avalue of 0.008 in PBS, but its a value increases 9-fold and 40-fold inthe viscous solvents octanol and glycerol, respectively. A 21-foldenhancement in the a value of camptothecin is observed upon binding ofdrug to vesicles composed of either DMPC or DMPG. Because of thesensitivity of a of the camptothecin drugs to membrane associations, themethod of fluorescence anisotropy titration was employed to study theequilibrium binding of camptothecin analogs with lipid bilayers. Asdescribed previously, the experiment consisted of determining the avalues for a set of samples where the drug concentration in each washeld constant (typically 1 or 2 μM), while the lipid concentration amongthe members of a set was varied from 0 to 0.29 M.

As a consequence of the brilliant fluorescence emissions from the newlysynthesized camptothecins (a summary of the spectral parameters can befound in Table 2), the adsorption isotherms summarized in FIGS. 16through 18 were relatively free from any background signal. Using drugconcentrations of 1 μM and long pass filters to isolate emitted lightfrom background signal (that is, scattered exciting light and extraneousfluorescence signal due to the possible presence of impurities), signallevels from drugs dissolved in PBS buffer were typically 99.97% in theabsence of membrane and greater than 98% in the presence of membrane.Adsorption isotherms were used to determine overall associationconstants for the camptothecin drugs. Overall association constants aredefined as:

    K=[A.sub.B ]/[A.sub.F ][L]

[A_(B) ] represents the concentration of bound drug, [A_(F) ] representsthe concentration of free drug, and [L] represents the total lipidconcentration in the vesicle suspension. This equation is valid when theconcentration of free lipid is approximately equal to the concentrationof total lipid (that is, the concentration of free lipid is insignificant excess over the concentration of bound drug). Provided thiscondition is satisfied, K may be determined from the inverse of theslope of a double reciprocal plot. In such a double reciprocal plot(representative data are shown in FIGS. 19 and 20), 1/fraction of thetotal drug bound is plotted vs. 1/lipid concentration, with ay-intercept value of 1 (for a system displaying binding sitehomogeneity). Such double-reciprocal plots for the associations of thenew camptothecin analogs with DMPC and DMPG small unilamellar vesicle(SUV) preparations were linear with good correlation coefficients. Thelinearity of these plots, as well as the corresponding plots for drugassociations with other types of membrane preparations, indicates thatfluorophore binding at these lipid concentrations is adequatelydescribed by the above equation.

The studies summarized in Table 3 examine the structural basis ofcamptothecin associations for lipid bilayers. Two types of membrane wereincluded in these studies which were conducted under near physiologicalconditions of pH and temperature; these membranes include fluid-phaseand electroneutral L-α-dimyristoylphosphatidylcholine (DMPC); andfluid-phase and negatively-charged L-α-dimyristoylphosphatidylglycerol(DMPG). DMPC and DMPG have identical chain length but the charge ontheir head groups differ.

                  TABLE 3                                                         ______________________________________                                        Overall association constants for camptothecin analogs                          interacting with unilamellar vesicles of electroneutral DMPC,                 negatively- charged DMPG in PBS buffer at PH 7.4 and 37° C.                                  K.sub.DMPC                                                                             K.sub.DMPG                                     Compound (M.sup.-1) (M.sup.-1)                                              ______________________________________                                        20(S)-camptothecin  100      100                                                7-methyl-20(S)-camptothecin 150 180                                           7-ethyl-20(S)-camptothecin 250 300                                            7-propyl-20(S)-camptothecin 540 600                                           7-methyl-10-methoxycamptothecin 220 200                                       7-ethyl-10-methoxycamptothecin 340 330                                        7-propyl-10-methoxycamptothecin 440 570                                       7-methyl-10-hydroxycamptothecin 220  90                                       7-ethyl-10-hydroxycamptothecin 260 160                                        7-propyl-10-hydroxycamptothecin 550 250                                       7-butyl-10-hydroxycamptothecin 2100  1270                                     DB-67 2700  2800                                                              DB-172 10500  10600                                                           DB-172 (Carboxylate form) 385 155                                             DB-173 5800  5800                                                             DB-174 9000  6600                                                             DB-174 (Carboxylate form) 540  60                                             CHJ-792 820 360                                                             ______________________________________                                    

In the studies of Table 3, binding isotherms were constructed using themethod of fluorescence anisotropy titration, and K values weredetermined from the slops of double-reciprocal plots. The K values aresubject to 10% uncertainty. Overall, the most striking feature of thedata contained in Table 3 is the strong modulation which can be achievedthrough either a sole substitution at the 7 position or dualsubstitution at the 7 and 10 positions. Included in Table 3 arepreviously known camptothecin compounds. Data for these agents wereincluded to show the highly lipophilic nature of the new camptothecinsrelative to the previous compounds. Topotecan was found to have a Kvalue for DMPC liposomes some 10 times less than that for camptothecin.From Table 3 it is clear that the compounds of the present invention aremuch more lipophilic than either camptothecin or topotecan. For example,the affinities of DB67 for membranes composed of DMPC or DMPG are27-fold and 28-fold greater that the corresponding values forcamptothecin. DB172 and DB174 are some 100-fold and 90-fold more apt tobind DMPC membranes when compared with camptothecin. DB173 is alsohighly lipophilic, displaying a K value for DMPC some 58-fold greaterthan that observed for camptothecin. In summary, the novel compounds ofthe present invention listed in Table 3 were found to display thehighest membrane affinities by far when compared against other, previouscamptothecin analogs containing the same α-hydroxy-β-lactone ringsystem.

Comparison of the Behavior of the Highly Lipophilic Camptothecins inAqueous Solution

FIG. 21 summarizes the stability of DB172 in phosphate buffered saline(PBS) buffer, pH 7.4, at physiological temperature. Shown in the figureare plots of lactone fraction as a function of time for DB172 atdifferent concentrations. Drug was added to solution from a concentratedDMSO stock solution such that the volumes of DMSO were very small (lessthan 0.5%) relative to the volume of water. The drug stability was foundto be markedly dependent on the drug concentration added. At the moredilute drug concentrations the drug hydrolyzes as previously observedfor other camptothecins containing the α-hydroxy-β-lactone moiety. Athigh drug concentrations marked stabilization of the lactone ring ofDB-172 was observed, a finding which is not typically observed for othercamptothecins.

FIG. 22 summarizes the dependence of the fluorescence intensity of DB172as a function of time and pH. In these experiments DB172 is added tosolution as the lactone form. At low pH where the drug remains in thelactone form, the change in intensity with time is the lowest. At pH 10,where the conditions are such that the lactone more readily hydrolyzesand forms carboxylate, a significant change in fluorescence intensity isobserved. It appears that a pH 10 nonfluorescent micellular aggregatescomposed of lactone disassemble and form open-ring carboxylate formsthat tend to exist in solution as monomeric fluorescent species.

FIG. 23 explores the fluorescence intensity of DB172 as a function ofconcentration. Following the addition of low concentration of lactonedrug to solution, the change in fluorescence signal is the greatestwhereas at high drug concentration (10 μM) the fluorescence intensitychanges are minimal. It is believed that a low concentration themicellular aggregates of DB172 displaying reduced fluorescence candisassemble and form fluorescent carboxylate species, but at higher drugconcentration the equilibrium favors that the agent remains in theaggregated or reduced fluorescence state. FIG. 24 shows that when thecarboxylate form of DB172 is added to solution at pH 10, no change influorescence signal is observed at pH 10 while at lower pH values wherelactone can form the fluorescence intensity decreases with time. Onceagain this decrease in fluorescence that occurs at reduced pH appear tobe due to the formation of lactone aggregates of reduced fluorescencequantum yield.

FIGS. 21 through 24 are consistent with the unusual ability of DB172lactone to self-associate and form micelles at micromolar drugconcentrations. The micellular DB172 aggregates display a reducedfluorescence. If conditions allow for hydrolysis to occur such thatcarboxylate forms, there is an increase in the fluorescence intensity ofthe sample. FIG. 25 compares the relative change in the fluorescenceemission of a sample following addition of lactone drug forms tosolution. In these experiments the values for each drug are normalizedto a value of 1 at time equals to zero, and the ability of the variousanalogs to disassociate in the event the drugs are aggregated at time=0is monitored with time. For several of the agents, hydrolysis to formcarboxylate occurs in the solution. Since the carboxylate forms are lesslikely to self-associate and exhibit reduced fluorescence, disruption ofthe aggregates by drug hydrolysis proceeds with an increase influorescence intensity. While DB172 self-associates to an exceptionallyhigh degree at time=0, the other agents self-associate to a much lesserextent and hence their signals are more constant with time and not assensitive to hydrolysis reactions. DB173 and DB67 appear to be much morelikely to be found in solution as monomeric drug relative to DB172. Thiscould be a favorable characteristic in that solutions for administrationto a patient will be more homogeneous than the aggregated DB172 particlesuspensions.

Markedly Enhanced Stabilities of Highly Lipophilic Camptothecins inHuman Blood

In was also demonstrated that the active lactone forms of highlylipophilic camptothecins of the present invention also persist for muchlonger times in human tissues such as blood when compared withwater-soluble analogs. FIG. 26 compares the stabilities of several newcamptothecin analogs in their free form in PBS buffer (Panel A) versusin whole blood (Panel B). These compounds include:7-t-butyldimethylsilylcamptothecin (DB202),7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB67),7-(3-chloropropyl)dimethylsilylcamptothecin (DB148), and7-(3-chloropropyl)-dimethylsilyl-10-hydroxycamptothecin (DB158). FIG. 27summarizes the stability data for DB172, DB173 and DB174 in PBS bufferonly (Panel A), PBS buffer containing physiologically relevant 30 mg/mllevels of HSA (Panel B), and human blood (Panel C). All experiments werecarried out at physiological temperature.

The highly biologically-active and lipophilic compound,7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67), was found todisplay superior stability in human blood, with a t_(1/2) of 130 min anda % lactone at equilibrium value of 30 (compare with % lactone atequilibrium values in whole human blood for 9-aminocamptothecin (<0.3%),camptothecin (5.3%), topotecan (8.6%), CPT-11 (21.0%), and SN-38(19.5%)). The stability data are summarized in Table 4. The new DB67agent was found to be 25-times more lipophilic than camptothecin, andits 10-hydroxy functionality was found to markedly aid in promotingstability in the presence of HSA. DB67 may be an ideal candidate for thetreatment of brain cancer. With intrinsic activity several-fold greaterthan camptothecin, DB67 displays very high equilibrium lactone levels inhuman blood, is not tightly bound to human albumin like camptothecin and9-aminocamptothecin, and is highly lipophilic which should enable theagent to more readily cross the blood brain barrier.

                  TABLE 3                                                         ______________________________________                                        Stability parameters for Camptothecin analogs in different biological         fluids                                                                             DRUG NAME    t.sub.1/2  % Lactone at                                       and FLUID (minutes) Equilibrium                                             ______________________________________                                        DB 202                                                                          Whole Blood 71.9 +/- 4.2  1.0 +/- 0.1                                         HSA 46.3 +/- 1.0  0.1 +/- 0.2                                                 PBS 27.9 +/- 1.9 12.2 +/- 0.4                                                 RBC 79.4 +/- 3.3 59.4 +/- 0.0                                                 DB 148                                                                        Whole Blood 85.6 +/- 9.2  2.2 +/- 3.6                                         HSA 16.0 +/- 0.2  2.7 +/- 0.2                                                 PBS 23.3 +/- 2.4 12.0 +/- 0.5                                                 RBC 59.7 +/- 2.9 43.4 +/- 0.8                                                 DB 67                                                                         Whole Blood 133.0 +/- 15.9 30.5 +/- 1.9                                       HSA 119.0 +/- 5.3  10.5 +/- 2.0                                               PBS 31.8 +/- 0.4 10.2 +/- 0.3                                                 RBC 51.4 +/- 0.5 41.4 +/- 0.7                                                 CHJ 792                                                                       Whole Blood 37.6 +/- 6.2 12.3 +/- 0.0                                         HSA 32.0 +/- 1.3  3.7 +/- 0.3                                                 PBS 31.3 +/- 0.6 10.5 +/- 0.3                                                 DB 158                                                                        Whole Blood 65.3 +/- 9.0 17.8 +/- 2.0                                         HSA 48.0 +/- 0.9 17.2 +/- 0.7                                                 PBS 29.5 +/- 1.7 11.3 +/- 1.8                                                 RBC 67.7 +/- 8.5 50.2 +/- 1.7                                                 7 TMS                                                                         Whole Blood 54.6 +/- 3.6 23.7 +/- 0.0                                         HSA 58.5 +/- 6.8 16.4 +/- 1.9                                                 PBS 34.6 +/- 1.0 11.1 +/- 0.1                                                 SN 38                                                                         Whole Blood 50.7 +/- 1.4 20.2 +/- 1.9                                         HSA 88.1 +/- 2.9 24.3 +/- 0.7                                                 Topotecan                                                                     Whole Blood 30.9 +/- 1.4 8.61 +/- 0.4                                         HSA 22.1 +/- 0.7 7.06 +/- 0.3                                                 DB 172                                                                        Whole Blood 106.6 +/- 10.8 24.1 +/- 2.6                                       HSA  86.1 +/- 10.3  4.3 +/- 1.0                                               DB 173                                                                        Whole Blood 69.0 +/- 4.0 36.3 +/- 1.7                                         HSA 49..0 +/- 1.3 15.5 +/- 0.5                                                PBS 45.3 +/- 2.0 11.9 +/- 0.3                                                 DB 174                                                                        Whole Blood 40.3 +/- 3.2 33.0 +/- 0.4                                         HSA 37.7 +/- 0.9 20.4 +/- 0.2                                                 PBS 29.7 +/- 0.4 13.7 +/- 0.7                                                 DB 124                                                                        Whole Blood 63.1 +/- 7.4 31.8 +/- 0.8                                         HSA 40.0 +/- 0.3 12.2 +/- 0.3                                                 PBS 30.3 +/- 1.0  8.9 +/- 0.3                                                 DB 104                                                                        Whole Blood 77.2 +/- 1.8 48.1 +/- 2.0                                         HSA 29.6 +/- 0.8  8.5 +/- 0.2                                                 PBS 24.1 +/- 1.0  7.5 +/- 0.2                                                 Camptothecin                                                                  Whole Blood 21.6 +/- 2.6  5.3 +/- 0.6                                         HAS 11.9 +/- 0.3 <0.5                                                         PBS 23.8 +/- 1.3   17 +/- 2.0                                               ______________________________________                                    

Also noteworthy are the very high human blood stabilities of DB-173 (36%lactone at equilibrium) and DB174 (33% lactone at equilibrium). Thesevalues are significantly greater than clinically relevant water-solublecamptothecins and they compete favorably with lipophilic camptothecinssuch as DB-172 that contain an unsubstituted A-ring.

Highly Lipophilic Camptothecins Display Oral Bioavailibility

FIG. 28 contains data which demonstrate that DB-67 is absorbed from thegastrointestinal tract. To evaluate the blood levels achieved followingdosing at 5 mg/kg, four animals were given 0.1 ml intrastomachinjections of DB-67 dissolved in DMSO. The stock contained DB67 at aconcentration of 1.6 mg/ml. At time points of 30 min, 1 hr, 2 hr, and 4hr, 1 ml samples of blood were drawn and collected by heart puncturing.The samples were centrifuged for 3 min and stored frozen until analysiswas carried out. Note that DB67 levels rise above the 40 ng/ml levelwith much of the drug persisting as active lactone drug. Thus, the novelhighly lipophilic analogs described here may be administered orally andwill appear in the bloodstream following administration.

Highly Lipophilic Camptothecins Display High Anticancer Potency Even inthe Presence Human Serum Albumin

As discussed previously, the spontaneous hydrolysis of camptothecin inaqueous solution yields the ring-opened carboxylate form which is farless active than the ring-closed lactone form. Therefore, theequilibrium between the lactone and carboxylate forms is a mostimportant determinant of the drug activities. Previous studies indicatedthat human serum albumin (HSA) greatly affects the equilibrium in favorof the carboxylate for camptothecin by preferentially interacting withthe carboxylate form. Because of this HSA effect, levels of thebiologically-active lactone form of camptothecin can be attenuated atthe tumor site. The drug-HSA interactions can be manipulated by drugstructural modification: A 10-OH substitution decreases the affinity ofdrug for HSA approximately 20-fold and an additional 7-ethylsubstitution further alters the binding in favor of the lactone form. Todetermine the impact of HSA on the cytotoxicities of the blood-stablecamptothecin analogs of the present invention, the cytotoxic effects ofthe camptothecin analogs were studied.

Sulphorhodamine B (SRB) assay was used. This assay measures the totalprotein levels in the living cells. Proteins from dead cells are lysedand removed in the washing step before TCA fixation. However, it ispossible that cells in the early stage of death still have theirmembrane integrity and therefore retain the protein contents inside. Asa result, the optical density at 490 nm can sometimes be overestimatedand the cytotoxicity underestimated. To validate the SRB assay, adiverse range of chemotherapeutic agents have been tested acrossmultiple panels of tumor cell lines, and close correlations have beenfound with standard tetrazolium (MIT) assay and clonogenic assays. TheSRB assay is now a well regarded assay and was recently approved by NCIas a standard assay for anticancer drug screening.

The cytotoxicities of various camptothecins against MDA-MB-435tumorigenic metastatic human breast cancer cells in the absence andpresence of 1 mg/ml HSA are summarized in Table 5. The cytotoxicityvalues for cells exposed to drug for 72 hrs. are summarized in Table 5.Overall, HSA is able to strongly attenuate the IC₅₀ values ofcamptothecin, but the extent to which HSA modulates the cytotoxicitiesof the new highly lipophilic analogs is significantly reduced. In fact,1 mg/ml HSA had no effect on the cytotoxic activity of DB173. In thepresence of HAS, DB173 displays a low nM potency against the humanbreast cancer cells. The ability of the agents to remain potent even inthe presence of albumin is potentially significant because of the greatabundance of this protein throughout the blood and tissue of the body.

                  TABLE 4                                                         ______________________________________                                        IC.sub.50 Values of Camptothecin and Analogs Against                            MDA-MB-435 Tumorogenic Metastatic Human Breast Cancer Cells                   in the Absence and Presence of Human Serum Albumin                                                  IC.sub.50 (nM)                                                                            IC.sub.50 (nM)                              Compound (w/o HSA) (w/HSA)                                                  ______________________________________                                        Camptothecin         8          >200                                            7-Ethyl-10-Hydroxycamptothecin (SN-38) 20 --                                  DB-174 12 --                                                                  DB-67  7 22                                                                   DB-173  4  4                                                                ______________________________________                                    

The present inventors have thus discovered that introduction of a silylgroup or a silylyalkyl group (for example, a trimethylsilyl group or atrimethylsilylethyl group) at position 7 of the camptothecin structuretypically results in a compound with better anti-tumor activity andhuman blood stability than camptothecin (see, for example, the compoundof Example 1 as compared to (20S)-CPT) and other previous camptothecinanalogs. Dual substitution at the 7 and 10 positions is even morefavorable (see, for example, compounds DB-173 and DB-174). The silylgroup or the silylalkyl group is also beneficial in the irinotecanseries (see, for example, the compound of Example 6 as compared toirinotecan).

The anti-tumor activity remains essentially unchanged when a hydroxygroup is introduced at position 10 of the compound of Example 1 toproduce the compound of Example 5. The compound of Example 6 is arelative of SN-38, the active metabolite of irinotecan. High activitieswere also observed in the present studies when a trimethylsilyl groupwas introduced in conjunction with a fluoro atom at position 11 (see,for example, the compound of Example 7), or a primary amine group atpositions 10 or 11 (see, respectively, Examples 8 and 9). Introductionof a fluoro atom in position 12 also results in an analog onlyapproximately 2 times less potent than camptothecin (see, Example 11 ascompared to (20S)-CPT). This result is surprising considering the pooractivity of the 12-substituted camptothecins reported previously in theliterature.

The novel camptothecin analogs of the present invention have uniquebiophysical and physiological properties. These highly lipophiliccamptothecin analogs with B-ring modifications and A- and B- ringmodifications display markedly improved α-hydroxy-δ-lactone ringstability in human blood. The camptothecin analogs of the presentinvention also display oral bioavailibility and potent anticanceractivity even in the presence of human serum albumin.

A mammal (human or animal) may thus be treated by a method whichcomprises the administration to the mammal of a pharmaceuticallyeffective amount of a compound of formula (1) or a pharmaceuticallyacceptable salt thereof. The condition of the mammal can thereby beimproved.

The compounds of the present invention can be administered in a varietyof dosage forms including, for example: parenterally (for example,intravenously, intradermally, intramuscularly or subcutaneously); orally(for example, in the form of tablets, lozengers, capsules, suspensionsor liquid solutions); rectally or vaginally, in the form of asuppository; or topically (for example, as a paste, cream, gel orlotion).

Optimal dosages to be administered may be determined by those skilled inthe art and will vary with the particular compound of formula (1) to beused, the strength of the preparation, the mode of administration, thetime and frequency of administration, and the advancement of thepatient's condition. Additional factors depending on the particularpatient will result in the need to adjust dosages. Such factors includepatient age, weight, gender and diet. Dosages may be administered atonce or divided into a number of smaller doses administered at varyingintervals of time.

EXAMPLES

The following examples are provided for illustration of the inventionand are not intended to be limiting thereof.

EXAMPLE 1

Preparation of (20S)-7-trimethylsilylcamptothecin ##STR11## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(3-trimethylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

To a solution of(S)-4-ethyl-4-hydroxy-6-iodo-3-oxo-1H-pyrano[3,4-c]-8-pyridone[iodopyridone (2), 250 mg, 0.746 mmol] in DME (2.5 mL) and DMF (0.60 mL)at 0° C. under argon was added 60% NaH in mineral oil (31.3 mg, 0.783mmol). LiBr (150 mg, 1.75 mmol) was added 10 min latter. After 15 min atroom temperature, 3-trimethylsilyl-2-propynyl bromide (430 mg, 2.24mmol) was injected and the reaction mixture was heated in the dark at65° C. for 20 h. The final solution was poured into brine (20 mL),extracted with AcOEt (6×15 mL) and dried (Na₂ SO₄). The residue obtainedafter removal of the solvents was subjected to flash-chromatography(CHCl₃ /AcOEt 95:5) to give 283 mg (85%) of a foam: [α]²⁰ _(D) +36.7 (c1, CHCl₃); IR (neat, cm⁻¹) 3384, 2940, 2166, 1730, 1634, 1518, 1406,1130, 841, 752; ¹ H NMR (300 MHz, CDCl₃) δ0.14 (s, 9H), 0.95 (t, J=7.4Hz, 3H), 1.77 (m, 2H), 3.66 (s, 1H), 5.00 (d, J=17.2 Hz, 1H), 5.10 (d,J=16.4 Hz, 1H), 5.15 (d, J=17.2 Hz, 1H), 5.49 (d, J=16.4 Hz, 1H), 7.16(s, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ-0.40, 7.7, 31.5, 44.5, 66.3, 71.8,90.9, 97.9, 116.5, 118.1, 148.6, 157.9, 173.3; HRMS (EI) m/z calcd forC₁₆ H₂₀ INO₄ Si M⁺) 445.0206, found 445.0203; LRMS (EI) m/z 445 M⁺),430, 416, 386.

(2) (20S)-7-Trimethylsilylcamptothecin

A solution of the compound prepared in (1) (36.6 mg, 0.082 mmol), phenylisonitrile (0.25 mmol) and hexamethylditin (42 mg, 0.123 mmol) inbenzene (1.3 mL) under argon was irradiated at 70° C. with a 275 W GEsunlamp for 10 h. The final reaction mixture was concentrated andsubjected to flash-chromatography (CHCl₃ /MeOH 96:4) to provide 18.8 mg(54%) of a slightly yellow solid: [α]_(D) ²⁰ +39.0 (c 0.2, CHCl₃ /MeOH4:1); ¹ H NMR (300 MHz, CDCl₃ /CD₃ OD 3:1) δ0.50 (s, 9H), 0.83 (t, J=7.4Hz, 3H), 1.74 (m, 2H), 3.72 (br s, 1H), 5.12 (d, J=16.4 Hz, 1H), 5.16(br s, 2 H), 5.47 (d, J=16.4 Hz, 1H), 7.49 (t, J=8.1 Hz, 1H), 7.54 (s,1H), 7.62 (t, J=8.1 Hz, 1H), 8.02 (d, J=8.1 Hz, 1H), 8.07 (d, J=8.1 Hz,1H); ¹³ C NMR (75 MHz, CDCl₃ /CD₃ OD 3:1) δ0.9, 7.2, 29.3, 31.0, 51.7,65.5, 98.3, 118.4, 127.3, 128.0, 129.7, 130.0, 131.8, 134.3, 144.7,145.6, 147.3, 151.1, 173.5; HRMS (EI) m/z calcd for C₂₃ H₂₄ N₂ O₄ Si(M⁺) 420.1505, found 420.1501; LRMS (EI) m/z 420 (M⁺), 391, 376, 361,347, 320, 291.

EXAMPLE 2

Preparation of (20S)-7-tert-butyldimethylsilylcamptothecin ##STR12## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(3-tert-butyldimethylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure described in Example 1-(1), iodopyridone (2)(200 mg, 0.60 mmol) and 3-tert-butyldimethylsilyl-2-propynyl bromide(280 mg, 1.20 mmol) provided, after flash-chromatography (CH₂ Cl₂ /AcOEt9:1), 173 mg (59%) of a white foam: [α]_(D) ²⁰ +58 (c 0.2, CHCl₃); IR(CHCl₃, cm⁻¹) 3548, 2950, 2927, 2859, 1745, 1648, 1526; ¹ H NMR (300MHz, CDCl₃) δ0.08 (s, 6H), 0.92 (m, 12H), 1.79 (m, 2H), 3.77 (br s, 1H),5.00-5.25 (m, 3H), 5.50 (d, J=16.4 Hz, 1H) 7.19 (s, 1H); ¹³ C NMR (75MHz, CDCl₃) δ-4.9, 7.63, 16.6, 26.0, 31.6, 44.5, 66.3, 71.8, 89.4, 98.6,100.0, 116.5, 118.1, 148.6, 158.0, 173.2; HRMS (EI) m/z calcd for C₁₉H₂₆ INO₄ Si (M⁺) 487.0679, found 487.0676; LRMS (EI) m/z 487 (M⁺), 430,386, 96, 81, 57.

(2) (20S)-7-tert-butyldimethylsilylcamptothecin

Following the procedure described in Example 1-(2), the compoundprepared in (1) (48.7 mg, 0.10 mmol) afforded, afterflash-chromatographies (CH₂ Cl₂ /MeOH 96:4; CH₂ Cl₂ /acetone 9:1), 24.8mg (54%) of an off yellow solid: [α]_(D) ²⁰ +35.5 (c 0.2, CHCl₃); IR(CHCl₃, cm⁻¹) 3028, 2980, 2960, 2932, 2859, 1741, 1658, 1600, 1555,1257, 1198, 1158, 1045; ¹ H NMR (300 MHz, CDCl₃) δ0.69 (s, 6H), 0.98 (s,9 H), 1.03 (t, J=7.3 Hz, 3H), 1.86 (m, 2H), 3.86 (s, 1H), 5.29 (d,J=16.3 Hz, 1H), 5.31 (s, 2H), 5.73 (d, J=16.3 Hz, 1H), 7.60 (t, J=6.3Hz, 1H), 7.60 (t, J=7.0 Hz, 1 H), 7.66 (s, 1H), 7.74 (t, J=7.3 Hz, 1H)8.20 (t, J=8.1 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃) δ-0.56, 7.80, 19.2,27.1, 31.6, 52.4, 66.3, 72.8, 97.7, 118.2, 127.0, 129.5, 129.6, 130.8,132.7, 136.0, 143.0, 146.4, 148.0, 150.1, 150.6, 157.4, 173.9; HRMS (EI)in/z calcd for C₂₆ H₃₀ N₂ O₄ Si (M⁺) 462.1974, found 462.1975; LRMS (EI)m/z 462 (M⁺), 450, 361, 331, 304, 245, 223, 57.

EXAMPLE 3

Preparation of (20S)-7-tert-butyldiphenylsilylcamptothecin ##STR13## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(3-tert-butyldiphenylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure described in Example 1-(1), iodopyridone (2)(200 mg, 0.60 mmol) and 3-tert-butyldiphenylsilyl-2-propynyl bromide(428 mg, 1.20 mmol) provided, after flash-chromatography (CH₂ Cl₂ /AcOEt9:1), 258 mg (70%) of a white foam: [α]_(D) ²⁰ +45.1 (c 0.2, CHCl₃); IR(CHCl₃, cm⁻¹) 3546, 2928, 2855, 1741, 1658, 1526; ¹ H NMR (300 MHz,CDCl₃) δ0.97 (t, J=7.3 Hz, 3H), 1.08 (s, 9H), 1.80 (m, J=7.1 Hz, 2H),3.76 (br s, 1H), 5.13 (d, J=16.4 Hz, 1H), 5.29 (d, J=2.5 Hz, 2H), 5.52(d, J=16.4 Hz, 1H), 7.22 (s, 1H), 7.32-7.40 (m, 6H), 7.76-7.78 (m, 4 H);¹³ C NMR (75 MHz, CDCl₃) δ7.6, 18.6, 27.0, 31.6, 44.6, 60.4, 66.3, 71.8,86.5, 99.9, 102.2, 116.6, 127.7, 129.6, 132.6, 135.6, 148.7, 157.8,173.2; HRMS (EI) m/z calcd for C₂₅ H₂₁ INO₄ Si (M-C₄ H₉ ⁺) 554.0279,found 554.0285; LRMS (EI) m/z 554 (M-C₄ H₉ ⁺), 587, 510, 220, 143, 105.

(2) (20S)-7-tert -butyldiphenylsilylcamptothecin

Following the procedure described in Example 1-(2), the compoundprepared in (1) (61.1 mg, 0.10 mmol) yielded, afterflash-chromatographies (CH₂ Cl₂ /MeOH 96:4; CH₂ Cl₂ /acetone 9:1), 26.5mg (45%) of a light yellow solid: [α]²⁰ _(D) +35.2 (c 0.2, CHCl₃); IR(CHCl₃, cm⁻¹) 3003, 2984, 2969, 2958, 2935, 1741, 1658, 1599, 1555,1428, 1226, 1216, 1158, 1102; ¹ H NMR (300 MHz, CDCl₃) δ1.00 (t, J=7.3Hz, 3H), 1.44 (s, 9H), 1.84 (m, 2H), 3.75 (s, 1H), 4.21 (d, J=5.7 Hz,2H), 5.19 (d, J=16.3 Hz, 1H), 5.64 (d, J=16.3 Hz, 1H), 7.43 (m, 5H),7.51 (t, J=7.3 Hz, 2H), 7.62 (s, 1H), 7.69 (m, 5H), 8.10 (d, J=8.5 Hz,1H), 8.22 (d, J=8.2 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ7.9, 20.4, 30.2,31.6, 52.2, 66.4, 72.8, 97.5, 118.2, 126.3, 128.6, 129.8, 130.3, 130.7,131.9, 132.2, 134.6, 134.64, 136.4, 136.5, 138.1, 140.9, 146.2, 148.4,149.9, 151.3, 157.1, 174.1; HRMS (EI) m/z calcd for C₃₆ H₃₄ N₂ O₄ Si(M⁺) 586.2281, found 586.2288; LRMS (EI) m/z 586 (M⁺), 542, 529, 485,428, 407, 321, 181, 131, 69.

EXAMPLE 4

Preparation of (20S)-10-acetoxy-7-trimethylsilylcamptothecin (see FIG.3) ##STR14## (1) 4-Acetoxyphenyl isonitrile (14)

To a solution of 4-acetoxyformanilide (13) (358 mg, 1.0 mmol) in CH₂ Cl₂(10 mL) at 0° C. were successively added tetrabromomethane (0.70 g, 2.1mmol), triphenylphosphine (525 mg, 2.1 mmol), and triethylamine (320 mL,2.1 mmol), and the resulting mixture was refluxed in the dark for 3 h.After evaporation of the solvents, the crude was triturated inice-cooled Et₂ O (20 mL) and filtered. The solvent was evaporated andthe residue was purified by flash-chromatography (hexanes/AcOEt 8:2) toafford 243 mg (76%) of a slightly brown solid: IR (neat, cm⁻¹) 2127,1768, 1501, 1370, 1201, 1180, 909; ¹ H NMR (300 MHz, CDCl₃) δ2.29 (s,3H), 7.11 (d, J=8.8 Hz, 2H), 7.38 (d, J=8.8 Hz, 2H); ¹³ C NMR (75 MHz,CDCl₃) δ21.0, 122.8, 127.6, 150.8, 164.3, 168.8; HRMS (EI) m/z calcd forC₉ H₇ NO₂ (M⁺) 161.0477, found 161.0474; LRMS (EI) m/z 161 (M⁺), 133,119, 91.

(2) (20S)-10-Acetoxy-7-trimethylsilylcamptothecin (15)

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (44.5 mg, 0.10 mmol) and the compound preparedin (1) (48.3 mg, 0.30 mmol) provided, after flash-chromatography (CHCl₃/acetone 10:1), 29.9 mg (63%) of a slightly yellow oil: [α]_(D) ²⁰ +29.9(c 0.5, CHCl₃); ¹ H NMR (300 MHz, CDCl₃) δ0.61 (s, 9H), 0.98 (t, J =7.4Hz, 3H), 1.86 (m, 2H), 2.38 (s, 3H), 4.13 (br s, 1 H), 5.24 (d, J=16.4Hz, 1H), 5.27 (s, 2H), 5.68 (d, J=16.4 Hz, 1H), 7.46 (dd, J=9.2, 2.5 Hz,1H), 7.60 (s, 1H), 7.96 (d, J=2.5 Hz, 1H), 8.13 (d, J=9.2 Hz, 1H); ¹³ CNMR (75 MHz, CDCl₃) δ1.4, 7.8, 21.4, 31.5, 51.7, 66.2, 97.6, 118.3,118.9, 124.6, 132.1, 135.0, 145.7, 146.1, 148.9, 150.1, 150.7, 157.3,169.1, 173.7; HRMS (EI) m/z calcd for C₂₅ H₂₆ N₂ O₆ Si (M⁺) 478.1560,found 478.1582; LRMS (EI) m/z 478 (M⁺), 436, 392, 377, 336, 277.

EXAMPLE 5

Preparation of (20S)-10-hydroxy-7-trimethylsilylcamptothecin (16)##STR15##

A solution of the compound (15) prepared in Example 5-(2) (16.8 mg,0.035 mmol) and K₂ CO₃ (9.6 mg, 0.070 mmol) in MeOH (100 mL) and H₂ O(100 mL) was stirred 1 h 30 at room temperature. The reaction mixturewas acidified with AcOH (2 drops), diluted with brine (10 mL) andextracted with AcOEt (10×10 mL). The combined organic layers were dried(Na₂ SO₄) and evaporated, and the residue was purified byflash-chromatographies (CHCl₃ /MeOH/AcOH 90:10:2; CHCl₃ /acetone 2:1) togive 15.1 mg (99%) of a white solid: [α]_(D) ²⁰ +18.9 (c 0.2, CHCl₃/MeOH 4:1); ¹ H NMR (300 MHz, CDCl₃ /CD₃ OD 4:1) δ0.45 (s, 9H), 0.84 (t,J=7.3 Hz, 3H), 1.75 (m, 2H), 5.12 (br s, 2H), 5.12 (d, J=16.3 Hz, 1H),5.48 (d, J=16.3 Hz, 1H), 7.24 (dd, J=9.1, 2.5 Hz, 1H), 7.39 (d, J=2.5Hz, 1H), 7.87 (d, J=9.1 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃ /CD₃ OD 4:1)δ0.8, 7.4, 31.1, 51.8, 65.7, 97.5, 109.8, 117.5, 122.3, 131.3, 133.7,134.6, 141.7, 142.6, 146.3, 147.5, 151.1, 156.3, 157.6; HRMS (EI) m/zcalcd for C₂₃ H₂₄ N₂ O₅ Si (M⁺) 436.1454, found 436.1450; LRMS (EI) m/z436 (M⁺), 392, 377, 336, 323.

Reaction of this compound with NH₂ CH₂ CH₂ NMe₂ followed by EtCOClprovided the open E-ring analog for biological testing.

EXAMPLE 6

Preparation of (20S)7-trimethylsilyl-irinotecan (see FIG. 6) ##STR16##(1) [1,4'] Bipieridinyl-1'-carboxylic acid 4-nitro-phenylester (32)

To a solution of 4-nitrophenyl chloroformate (31) (5.15 g, 25.6 mmol) in150 mL of dry THF at -78° C. was added triethylamine (10.7 mL, 76.2mmol), followed by a solution of 4-piperidinopiperidine (30) (4.51 g,25.6 mmol) in 40 mL of THF. This solution was stirred for two hours,after which the solvent was removed, and the residue was taken up inAcOEt, filtered and evaporated. The crude yellow solid was passedthrough a pad of neutral alumina using AcOEt as an eluent to yield,after evaporation, 6.73 g (79%) of a white solid: IR (CHCl₃, cm⁻¹) 3046,2937, 2859, 1704, 1620, 1513, 1466, 1242, 1197; ¹ H NMR (300 MHz, CDCl₃)δ1.20-1.80 (m, 8H), 1.90 (d, J=12.7 Hz, 2H), 2.20-2.70 (m, 5H), 2.87 (t,J=12 Hz, 1H), 3.01 (t, J=12 Hz, 1H), 4.30 (br s, 2H), 7.29 (d, J=9 Hz,2H), 8.26 (d, J=9 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃) δ24.6, 26.3, 27.5,28.2, 40.1, 44.4, 50.1, 62.0, 122.2, 124.9, 144.8, 151.9, 156.3; HRMS(EI) m/z calcd for C₁₇ H₂₃ N₃ O₄ (M⁺) 333.1676, found 333.1688; LRMS(EI) m/z 333 (M⁺), 195, 167, 124, 110, 96, 55.

(2) [1,4'] Bipiperidinyl-1'-carboxylic acid 4-amino-phenylester

To a solution of the compound prepared in (1) (1.012 g, 3.03 mmol) inAcOEt (125 ml) was added 10% Pd/C (0.15 g). The system was purgedseveral times with argon, and a 1 L balloon of H₂ was added. Afterstirring the resulting mixture at room temperature for 12 hours, thecatalyst was removed by filtration through celite and the solvent wasevaporated to give 835 mg (91%) of a white solid: IR (CHCl₃, cm⁻¹) 3453,3400, 3028, 2936, 2859, 1703, 1513, 1429, 1242, 1226, 1210, 1197; ¹ HNMR (300 MHz, CDCl₃) δ1.30-1.70 (m, 8H), 1.86 (d, J=12.6 Hz, 2H),2.33-2.62 (m, 5H), 2.68-3.04 (m, 2H), 3.58 (br s, 2H), 4.30 (br s, 2H),6.64 (d, J=6.0 Hz, 2H), 6.87 (d, J=6.0 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃)δ24.6, 26.3, 27.5, 28.1, 43.8, 43.9, 50.1, 62.3, 115.4, 122.3, 143.4,143.7, 154.1; HRMS (EI) m/z calcd for C₁₇ H₂₅ N₃ O₂ (M⁺) 303.1944 ,found 303.1947; LRMS (EI) m/z 303 (M⁺), 195, 167, 124, 108, 96, 80, 65,55.

(3) [1,4'] Bipiperidinyl-1'-carboxylic acid 4-formylamino-phenylester(33)

To a stirred solution of dicyclohexylcarbodiimide (272 mg, 1.32 mmol) inCH₂ Cl₂ (5 mL) at 0° C. was added 98% formic acid (60.7 mg, 1.32 mmol)dropwise. After 10 minutes, the resulting mixture was added via syringeto a solution of the compound prepared in Example (2) (200 mg, 0.66mmol) in pyridine (5 mL) at 0° C. The reaction mixture was then allowedto warm to room temperature and stirred 3 h. The pyridine solvent wasevaporated and the residue was taken up in CH₂ Cl₂, filtered, evaporatedand subjected directly to a basic alumina column (CH₂ Cl₂ /MeOH 95:5) togive 118 mg (83%) of a white solid, which consists, at room temperature,of a mixture of the cis and trans rotamers originating from hinderedrotation around the formamide carbon-nitrogen bond: IR (CHCl₃, cm⁻¹)3025, 3013, 2937, 2888, 2861, 1703, 1517, 1466, 1275, 1226, 1210; ¹ HNMR (300 MHz, CDCl₃) δ1.38-1.80 (m, 8H), 1.90 (d, J=12 Hz, 2H),2.40-2.70 (m, 5H), 2.83 (t, J=12 Hz, 1H), 2.97 (t, J=12 Hz, 1H), 4.32(m, 2H), 7.03-7.11 (m, 3H), 7.37 (br s, 0.5H) (cis), 7.46 (d, J=10 Hz,1H), 7.53 (d, J=11 Hz, 0.5H) (trans), 8.32 (d, J=2 Hz, 0.5H) (cis), 8.59(d, J=11 Hz, 0.5H) (trans); ¹³ C NMR (75 MHz, CDCl₃) δ24.6, 26.3, 27.6,28.1, 44.2, 44.0, 50.1, 82.2, 120.0, 121.0, 122.1, 123.0, 133.9, 134.3,147.5, 148.9, 153.9, 153.4, 159.1, 162.5; HRMS (EI) m/z calcd for C₁₈H₂₅ N₃ O₃ (M⁺) 331.1884, found 331.1896; LRMS (EI) m/z 331 (M⁺), 244,202, 167, 124, 80, 55.

(4) [1,4'] Bipiperidinyl-1'-carboxylic acid 4-isonitrilo-phenylester(34)

To a solution of the compound prepared in Example (3) (90.1 mg, 0.272mmol) in CH₂ Cl₂ (10 mL) were successively added triethylamine (69.5 mg,0.688 mmol) them dropwise, at 0° C., a solution of triphosgene (68 mg,0.229 mmol) in dry CH₂ Cl₂ (10 mL). The mixture was stirred 2 hours atroom temperature, washed with 7% NaHCO₃ (5 mL) and dried (MgSO₄). Thecrude brown residue obtained after evaporation of the solvent wassubjected to flash-chromatography (Et₂ O/Et₂ NH 95:5) to yield 67.2 mg(79%) of a white solid: IR (CHCl₃, cm⁻¹) 3034, 2937, 2131, 1718, 1504,1429, 1233, 1224, 1213, 1198, 1184; ¹ H NMR (300 MHz, CDCl₃) δ1.32-1.75(m, 8H), 1.90 (br d, J=12.4 Hz, 2H), 2.32-2.65 (m, 5H), 2.84 (t, J=12.3Hz, 1H), 2.98 (t, J=12.1 Hz, 1H), 4.20-4.40 (m, 2H), 7.14 (d, J=8.8 Hz,2H), 7.37 (d, J=8.8 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃) δ25.0, 26.5, 27.8,28.5, 44.4, 50.6, 62.7, 123.3, 127.8, 152.1, 153.1, 164.4; HRMS (EI) m/zcalcd for C₁₈ H₂₃ N₃ O₂ (M⁺) 313.1779, found 313.1790; LRMS (EI) m/z 313(M⁺), 195, 167, 124, 110, 84, 55.

(5) (20S)-7-Trimethylsilyl-Irinotecan (35)

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (44.5 mg, 0.10 mmol), the compound prepared in(4) (93.9 mg, 0.3 mmol), and hexamethylditin (50 mg, 0.15 mmol) in drybenzene (1.5 mL) were irradiated for 9 hours at 70° with a 275 W GEsunlamp. The reaction was evaporated, dissolved in MeOH with a few dropsof DMSO to aid solubility and injected into a Waters reverse phase HPLC.The conditions used to effect separation were as follows. A Waters 600Esystem controller with a Waters 490E Programmable multiwavelengthdetector, a Sargent Welch plotter and Waters C-18 25×10 cartridgecolumns were employed. A gradient elution, [5:95 MeCN/H₂ O (0.1% TFA) to30:70 MeCN/H₂ O (0.1% TFA)], over 40 minutes time at 20 mL/min gave asemipurified grey solid after lyophilization. The grey solid was furtherpurified (CH₂ Cl₂ /EtOH 70:30) on a chromatotron using a 1 mm plate togive 12 mg (19%) of a yellow solid: [α]_(D) ²⁰ +14.8 (c 0.2, CHCl₃); IR(CHCl₃, cm⁻¹) 3023, 2957, 2933, 1720, 1659, 1601, 1216, 1191, 1175,1158; ¹ H NMR (300 MHz, CDCl₃) δ0.64 (s, 9H), 1.03 (t, J=7.3 Hz, 3H),1.50-1.51 (br m, 2H), 1.51-1.52 (br m, 6H), 1.84 (m, J=7.3 Hz, 2H),2.01-2.10 (br m, 2H), 2.60-2.75 (br s, 5H), 2.75-3.12 (br m, 2H),4.30-4.50 (br m, 2H), 5.30 (d, J=16.3 Hz, 1H), 5.31 (s, 2H), 5.74 (d,J=16.3 Hz, 1H), 7.55 (dd, J=9.0, 2.4 Hz, 1H), 7.63 (s, 1H), 8.01 (d,J=2.3 Hz, 1H), 8.19 (d, J=9 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ1.5, 7.8,25.4, 29.7, 31.5, 43.8, 50.1, 51.8, 62.5, 66.3, 72.8, 97.5, 118.1,119.0, 125.1, 132.0, 132.3, 134.9, 143.4, 145.6, 146.4, 150.1, 150.5,152.8, 157.4, 174.0; HRMS (EI) m/z calcd for C₃₄ H₄₂ N₄ O₆ Si (M⁺)630.2898, found 630.2874; LRMS (EI) m/z 630 (M⁺), 586, 501, 457, 195,167, 153, 124, 111, 96, 84.

EXAMPLE 7

Preparation of (20S)-11-fluoro-7-trimethylsilylcamptothecin (see FIG. 2)##STR17## (1) 3-Fluoro-2-trimethylsilylbenzaldehyde (7)

The preparation of 3-fluoro-2-trimethylsilylbenzaldehyde proceedsthrough a selective ortho-metallation. See Comins, D. L. et al., J. Org.Chem., 49, 1078 (1984). See also Snieckus, V., Chem. Rev., 90, 879(1990). To a solution of N,N,N'-trimethylethylenediamine (2.70 mL, 20mmol) in THF (50 mL) was slowly added 1.6 N n-BuLi in hexanes (13 mL, 21mmol) at -20° C., followed by 3-fluorobenzaldehyde (2.10 mL, 20 mmol) 15min latter. After 15 minute at this temperature, 1.6 N n-BuLi in hexanes(38 mL, 60 mmol) was injected and the solution was stirred 1 h 30 at-35° C. Chlorotrimethylsilane (15 mL, 120 mmol) was added and thereaction mixture was stirred overnight at room temperature. The finalsolution was poured into ice-cooled 1 N HCl (150 mL), quickly extractedwith Et₂ O (3×100), washed with brine and dried (Na₂ SO₄). Afterevaporation of the solvents, the residue was purified byflash-chromatography (hexanes/AcOEt 95:5) to provide 3.25 g (83%) of anoil: IR (neat, cm⁻¹) 1701, 1440, 1252, 1233, 1109, 848, 764; ¹ H NMR(300 MHz, CDCl₃) δ0.40 (d, J=2.6 Hz, 9H), 7.18 (br t, J=9.0 Hz, 1H),7.47 (ddd, J₁ =J₂ =8.1 Hz, J₃ =5.4 Hz, 1H), 7.70 (br d, J=7.5 Hz, 1H);¹³ C NMR (75 MHz, CDCl₃) δ1.8, 120.8 (d, J_(CF) =29 Hz), 126.8, 128.2,131.2, 143.3, 167.6 (d, J_(CF) =244 Hz), 192.4; HRMS (EI) m/z calcd forC₉ H₁₀ FOSi (M-CH₃ ⁺) 181.0485, found 181.0482; LRMS (EI) m/z 181 (M-CH₃⁺), 151, 125, 103, 91.

(2) 3-Fluoro-2-trimethylsilylbenzoic acid

A classical oxidation to the free acid was then performed. See Hill, L.R. et al., J. Org. Chem., 50, 470 (1985). To a solution of the compoundprepared in (1) (3.41 g, 17.3 mmol) in tert-butanol (20 mL) weresuccessively added a 2 N solution of 2-methyl-2-butene in THF (55 mL,110 mmol) then slowly, over a period of 10 minutes, a solution of 80%NaClO₂ (2.55 g, 22.5 mmol) and NaH₂ PO₄.H₂ O (3.10 g, 22.5 mmol) inwater (18 mL). The resulting mixture was stirred 16 h at roomtemperature, the tert-butanol was evaporated, and the residue was takenup in 1 N NaOH (50 mL) and washed with hexanes (3×20 mL). The aqueouslayer was acidified with 1 N HCl to pH 2, saturated with NaCl, andextracted with Et₂ O (3×50 mL). The combined organic layers were dried(Na₂ SO₄) and evaporated to provide 3.13 g (85%) of a white solid: IR(NaCl, cm⁻¹) 2982, 1700, 1434, 1294, 1271, 1253, 1230, 849, 763; ¹ H NMR(300 MHz, CDCl₃) δ0.39 (d, J=2.6 Hz, 9H), 7.16 (br t, J=9.1 Hz, 1H),7.41 (ddd, J₁ =J₂ =7.9 Hz, J₃ =5.6 Hz, 1H), 7.73 (br d, J=7.7 Hz, 1H);¹³ C NMR (75 MHz, CDCl₃) δ1.3, 119.5 (d, J_(CF) =27 Hz), 126.0, 127.3,130.9, 138.0, 167.5 (d, J_(CF) =243 Hz), 174.5; HRMS (EI) m/z calcd forC₉ H₁₀ FO₂ Si (M-CH₃ ⁺) 197.0434, found 197.0433; LRMS (EI) m/z 197(M-CH₃ ⁺), 179, 133, 115, 105.

(3) 3-Fluoro-2-trimethylsilylphenyl isocyanate (8)

Preparation of the intermediate isocyanate was carried out via a Curtiusrearrangement. See Capson, T. L. et al., Tetrahedron Lett., 25, 3515(1984) and references herein. To a solution of the compound prepared in(2) (3.03 g, 14.3 mmol) in CH₂ Cl₂ (20 mL) was added oxalylchloride(1.30 mL, 15.0 mmol) and the resulting mixture was stirred 3 h at roomtemperature. The residue obtained after evaporation of the solvent wasdiluted with THF (10 mL) and injected with vigorous stirring to aice-cooled solution of NaN₃ (3.70 g, 57 mmol) in H₂ O (20 mL) andacetone (50 mL). After 15 min at 0° C. and 1 min at room temperature,the solution was extracted with Et₂ O (4×50 mL) and dried (Na₂ SO₄). Theresidue obtained after evaporation of solvents was refluxed in toluenefor 1 h 30 to provide, upon solvent removal, 2.85 g (79%) of a slightlyyellow oil: IR (neat, cm⁻¹) 2269, 1598, 1433, 1252, 1228, 846, 788; ¹ HNMR (300 MHz, CDCl₃) δ0.38 (d, J=1.9 Hz, 9H), 6.82 (br t, J =8.3 Hz,1H), 6.90 (br d, J=8.2 Hz, 1H), 7.25 (ddd, J₁ =J₂ =8.1 Hz, J₃ =6.6 Hz,1H); ¹³ C NMR (75 MHz, CDCl₃) δ0.4, 112.6 (d, J_(CF) =26 Hz), 120.5,122.5, 131.5, 139.2, 167.4 (d, J_(CF) =241 Hz)

(4) 3-Fluoro-2-trimethylsilylphenyl isonitrile (9)

A deoxygenation then afforded the expected isonitrile. See Baldwin, J.E. et al., Tetrahedron , 39, 2989 (1983). Triethylamine (4.10 mL, 29.3mmol) was added slowly at 0° C. to a 2 N solution of trichlorosilane inCH₂ Cl₂ (8.40 mL, 16.8 mmol) followed, 5 min latter, by the compoundprepared in Example (3) (2.35 g. 11.2 mmol). After 1 h 30 at 0° C. and30 min at room temperature, the solution was saturated with NH₃,filtered over Celite, washed with 5% NaH₂ PO₄ and dried (Na₂ SO₄). Thecrude obtained after evaporation of the solvent was then subjected toflash-chromatography (hexanes/AcOEt 95:5) to afford 1.42 g (66%) of aslightly purple liquid: IR (neat, cm⁻¹) 2114, 1598, 1440, 1254, 1237,1110, 943, 848, 793; ¹ H NMR (300 MHz, CDCl₃) δ0.45 (d, J=1.8 Hz, 9H),7.01 (br t, J=8.3 Hz, 1H), 7.17 (br d, J=7.7 Hz, 1H), 7.32 (ddd, J₁ =J₂=8.0 Hz, J₃ =6.1 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ0.1, 116.5 (d,J_(CF) =26 Hz), 124.3, 131.6, 166.8 (d, J_(CF) =243 Hz), 166.9; HRMS(EI) m/z calcd for C₁₀ H₁₂ FNSi (M⁺) 193.0723, found 193.0715; LRMS (EI)m/z 193 (M⁺), 178, 150, 116, 105.

(5) (20S)-11-Fluoro-7,12-bis(trimethylsilyl)camptothecin (11)

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (43.5 mg, 0.098 mmol) and the compoundprepared in Example (4) (76 mg, 0.39 mmol) provided, afterflash-chromatography (CHCl₃ /acetone 20:1), 33.4 mg (67%) of a slightlyyellow oil: [α]_(D) ²⁰ +23.6 (c 0.2, CHCl₃) ; ¹ H NMR (300 MHz, CDCl₃)δ0.53 (d, J=1.7 Hz, 9H), 0.60 (s, 9H), 1.02 (t, J=7.4 Hz, 3H), 1.88 (m,2H), 3.82 (br s, 1H), 5.28 (d, J=16.3 Hz, 1H), 5.29 (br s, 2H), 5.72 (d,J=16.3 Hz, 1H), 7.31 (t, J=8.7 Hz, 1 H), 7.46 (s, 1H), 8.18 (dd, J=9.2,5.9 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ1.6, 1.7, 7.7, 31.4, 51.8, 66.3,72.7, 97.2, 117.8 (d, J_(CF) =33 Hz), 124.3 (d, J_(CF) =28 Hz), 128.9,131.1, 133.1, 144.4, 146.7, 150.1, 153.4, 157.4, 167.6 (d, J_(CF) =245Hz), 173.9; HRMS (EI) m/z calcd for C₂₆ H₃₁ FN₂ O₄ Si₂ (M⁺) 510.1806,found 510.1806; LRMS (EI) m/z 510 (M⁺), 495, 466, 451, 395, 319.

(6) (20S)-11-Fluoro-7-trimethylsilylcamptothecin (12)

A solution of the compound prepared in Example (5) (19.5 mg, 0.038 mmol)in 48% HBr (1 mL) was heated at 50° C. for 20 h. The reaction mixturewas slowly poured with vigorous stirring into saturated NaHCO₃ (10 mL),extracted with AcOEt (6×20 mL) and dried (Na₂ SO₄). After evaporation ofthe solvent, the residue was purified by flash-chromatography (CHCl₃/acetone 8:1) to give 12.5 mg (83%) of a slightly yellow solid: [α]_(D)²⁰ +39.6 (c 0.2, CHCl₃); ¹ H NMR (300 MHz, CDCl₃) δ0.62 (s, 9H), 1.01(t, J=7.4 Hz, 3H), 1.87 (m, 2H), 3.81 (br s, 1H), 5.28 (d, J=16.4 Hz,1H), 5.28 (br s, 2H), 5.72 (d, J=16.4 Hz, 1H), 7.31 (ddd, J=9.6, 7.8,2.8 Hz, 1H), 7.61 (s, 1H), 7.78 (dd, J =9.7, 2.7 Hz, 1H), 8.19 (dd,J=9.4, 5.8 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ1.6, 7.8, 31.5, 51.7,66.3, 72.7, 97.8, 114.3 (d, J_(CF) =20 Hz), 117.7 (d, J_(CF) =26 Hz),118.5, 128.9, 130.0, 133.9, 144.4, 146.1, 149.3, 150.1, 151.7, 157.4,162.6 (d, J_(CF) =250 Hz), 173.9; HRMS (EI) m/z calcd for C₂₃ H₂₃ FN₂ O₄Si (M⁺) 438.1411, found 438.1412; LRMS (EI) m/z 438 (M⁺), 409, 394, 379,365, 338, 309.

EXAMPLE 8

Preparation of (20S)-10-amino-7-trimethylsilylcamptothecin (see FIG. 4)(CHJ-792) ##STR18## (1) 4-tert-Butyloxycarbonylaminophenyl isonitrile(18)

The isonitrile was prepared in 2 steps via classical Boc-protectionfollowed by dehydration. See Einhorn, J. et al., Synlett, 37 (1991). Amixture of 4-aminoformanilide (1.71 g, 12.6 mmol), di-tert-butyldicarbonate (2.87 g, 13.2 mmol) and NaHCO₃ (1.11 g, 13.2 mmol) inabsolute EtOH (50 mL) was sonicated in a cleaning bath for 4 h. Thefinal solution was filtered through a pad of Celite and concentrated todryness. The residue was taken up in half brine (50 mL), extracted withAcOEt (6×30 mL) and dried (Na₂ SO₄). After evaporation of the solvent,the residual oil was subjected to flash-chromatography (CHCl₃ /MeOH95:5) to give 2.85 g (96%) of 4-tert-butyloxycarbonylaminoformanilide,as a white solid. This intermediate (945 mg, 4.0 mmol) was subjected tothe conditions described in Example 5-(1) to provide, afterflash-chromatography (hexanes/AcOEt 9:1), 502 mg (58%) of a slightlybrown solid: IR (NaCl, cm⁻¹) 3370, 2121, 1691, 1524, 1412, 1364, 1239,1158, 832; ¹ H NMR (300 MHz, CDCl₃) δ1.48 (s, 9H), 6.75 (br s, 1H), 7.26(d, J=8.8 Hz, 2), 7.37 (d, J=8.8 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃)δ28.2, 81.3, 118.5, 127.1, 139.4, 152.3, 162.7; HRMS (EI) m/z calcd forC₁₂ H₁₄ N₂ O₂ (M⁺) 218.1055, found 218.1044; LRMS (EI) m/z 218 (M⁺),162, 144.

(2) (20S)-10-tert-Butyloxycarbonylamino-7-trimethylsilyl camptothecin(19)

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (44.5 mg, 0.10 mmol) and the compound preparedin Example (1) (65 mg, 0.30 mmol) provided, after flash-chromatography(CHCl₃ /acetone 6:1), 32.5 mg (60%) of a slightly yellow solid: [α]_(D)²⁰ +28.0 (c 0.2, CHCl₃); ¹ H NMR (300 MHz, CDCl₃) δ0.63 (s, 9H), 0.99(t, J=7.4 Hz, 3H), 1.53 (s, 9H), 1.86 (m, 2H), 4.03 (br s, 1H), 5.24 (d,J=16.2 Hz, 1H), 5.26 (s, 2H), 5.70 (d, J=16.2 Hz, 1H), 7.00 (br s, 1H),7.47 (dd, J=9.2, 2.3 Hz, 1H), 7.55 (s, 1H), 8.02 (d, J=9.2 Hz, 1H), 8.56(br s, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ1.3, 7.8, 28.2, 31.5, 51.8, 66.3,72.8, 97.1, 114.4, 117.8, 122.6, 131.3, 132.8, 135.0, 137.2, 142.9,144.3, 146.6, 149.2, 150.1, 157.4, 173.9; HRMS (EI) m/z calcd for C₂₃H₂₅ N₃ O₄ Si (M-Boc⁺) 435.1614, found 435.1612; LRMS (EI) m/z 535 (M⁺),479, 435, 391, 362, 335.

(3) (20S)-10-Amino-7-trimethylsilylcamptothecin (20)

A solution of the compound prepared in Example (2) (75.5 mg, 0.141 mmol)and TFA (500 mL) in CH₂ Cl₂ (2 mL) was stirred 3 h at room temperature.The reaction mixture was then poured into saturated NaHCO₃ (50 mL),extracted with AcOEt (10×15 mL) and dried (Na₂ SO₄). The residueobtained after evaporation of the solvents was purified byflash-chromatography (CHCl₃ /MeOH 95:5) to afford 55.4 mg (90%) of ayellow solid: [α]_(D) ²⁰ +18.7 (c 0.15, CHCl₃ /MeOH 4:1); ¹ H NMR (300MHz, CDCl₃ /CD₃ OD 4:1) δ0.40 (s, 9H), 0.80 (t, J=7.4 Hz, 3H), 1.70 (m,2H), 5.05 (s, 2H), 5.08 (d, J=16.3 Hz, 1H), 5.43 (d, J=16.3 Hz, 1H),7.05 (br s, 1H), 7.07 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 7.74 (d, J=8.0Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃ /CD₃ OD 4:1) δ0.6, 7.2, 30.8, 51.8,65.5, 72.7, 97.0, 107.2, 116.8, 122.0, 130.7, 134.0, 134.7, 139.9,141.7, 145.8, 146.9, 151.2, 157.5, 173.7; HRMS (EI) m/z calcd for C₂₃H₂₅ N₃ O₄ Si (M⁺) 435.1614, found 435.1613; LRMS (EI) m/z 435 (M⁺), 391,376, 335, 290.

EXAMPLE 9

Preparation of (20S)-11-amino-7-trimethylsilylcamptothecin ##STR19## (1)3-tert-Butyloxycarbonylaminophenyl isonitrile

The isonitrile was prepared in 2 steps following the same procedures asdescribed in Example 9-(1). In the first step, the Boc-protection of3-aminoformanilide (1.80 g, 13.2 mmol) provided, afterflash-chromatography (CHCl₃ /MeOH 95:5), 2.65 g (85%) of3-tert-butyloxycarbonylaminoformanilide, as a white solid. Thisintermediate (412 mg, 1.74 mmol) was then subjected to the conditionsdescribed in Example 5-(1) to provide, after flash-chromatography(hexanes/AcOEt 9:1), 190 mg (50%) of a brown solid: IR (NaCl, cm⁻¹)3318, 2126, 1715, 1603, 1547, 1433, 1236, 1162, 782; ¹ H NMR (300 MHz,CDCl₃) δ1.49 (s, 9H), 6.67 (br s, 1H), 7.00 (m, 1H), 7.20-7.30 (m, 2H),7.60 (br s, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ28.2, 81.3, 116.0, 118.9,120.6, 129.8, 139.5, 152.3, 163.6; HRMS (EI) m/z calcd for C₁₂ H₁₄ N₂ O₂(M⁺) 218.1055, found 218.1047; LRMS (EI) m/z 218 (M⁺), 196, 162, 152,118.

(2) (20S)-11-Amino-7-trimethylsilylcamptothecin

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (44.5 mg, 0.10 mmol) and the compound preparedin Example (1) (65.5 mg, 0.3 mmol) afforded, afterflash-chromatographies (CHCl₃ /MeOH 95:5; CHCl₃ /acetone 5:1), 23.1 mg(43%) of a slightly yellow oil. This intermediate (14.7 mg, 0.027 mmol)was then deprotected following the conditions described in Example 9-(3)to provide, after flash-chromatography (CHCl₃ /MeOH 9:1), 11.8 mg (99%)of (20S)-11-amino-7-trimethylsilylcamptothecin, as a yellow solid andwith the exclusion of other isomers: [α]_(D) ²⁰ +15.0 (c 0.1, CHCl₃/MeOH 4:1); ¹ H NMR (300 MHz, CDCl₃ /CD₃ OD 4:1) δ0.44 (s, 9H), 0.86 (t,J=7.4 Hz, 3H), 1.76 (m, 2H), 5.08 (s, 2H), 5.14 (d, J=16.4 Hz, 1H), 5.50(d, J=16.3 Hz, 1H), 6.97 (dd, J=9.2, 2.5 Hz, 1H), 7.07 (d, J=2.5 Hz,1H), 7.50 (s, 1H), 7.84 (d, J=9.2 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃ /CD₃OD 4:1) δ1.1, 7.4, 31.0, 51.7, 65.6, 97.9, 107.9, 117.8, 119.7, 125.9,127.1, 129.0, 130.4, 135.4, 144.3, 149.5, 149.9, 151.1, 157.6, 175.3;HRMS (EI) m/z calcd for C₂₃ H₂₅ N₃ O₄ Si (M⁺) 435.1614, found 435.1626;LRMS (EI) m/z 435 (M⁺), 406, 391, 376, 335.

EXAMPLE 10

Preparation of (20S)-11-fluoro-10-amino-7-trimethylsilylcamptothecin(see FIG. 5) ##STR20## (1)4-tert-Butyloxycarbonylamino-3-fluoro-1-nitrobenzene (22)

To a solution of 2-fluoro-4-nitroaniline (21) [prepared according toKatritsky, A. R. et al., J. Org. Chem., 51, 5039 (1986)] (2.16 g, 13.9mmol) in CH₂ Cl₂ (25 mL) were successively added di-tert-butyldicarbonate (3.19 g, 14.6 mmol), triethylamine (2.95 mL, 20.8 mmol) and4-dimethylaminopyridine (210 mg, 1.67 mmol) and the reaction mixture wasstirred 16 h at room temperature. The final solution was diluted withCH₂ Cl₂ (75 mL), washed with ice-cooled 5% citric acid (4×50 mL) anddried (Na₂ SO₄). After evaporation of the solvent, the residue wassubjected to flash-chromatography (Hexanes/AcOEt 9:5) to provide, inorder of elution, first 1.95 g (55%) of the mono-protected derivative,4-tert-butyloxycarbonylamino-3-fluoro-1-nitrobenzene, secondly 1.13 g(23%) of the bis-protected derivative,4-di-tert-butyloxycarbonylamino-3-fluoro-1-nitrobenzene. Thecharacteristics of the mono-protected derivative are as follows: ¹ H NMR(300 MHz, CDCl₃) δ1.52 (s, 9H), 6.99 (br s, 1H), 7.95 (m, 1H), 8.03 (brd, J=9.2 Hz, 1H), 8.34 (br t, J=8.5 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃)δ28.1, 82.5, 110.9 (d, J_(CF) =23 Hz), 118.3, 120.8, 133.5, 141.7, 150.1(d, J_(CF) =243 Hz), 151.4; HRMS (EI) m/z calcd for C₁₁ H₁₃ FN₂ O₄ (M⁺)256.0859, found 258.0854; LRMS (EI) m/z 256 (M⁺), 200, 182, 57.

(2) 4-tert-Butyloxycarbonylamino-3-fluoroaniline (24)

Reduction of the nitro group to the amine function was carried outfollowing a classical procedure. See Ram, S. et al., Tetrahedron Lett.,25, 3415 (1984). To a solution of the compound prepared in Example (1)(1.62 g, 6.32 mmol) and ammonium formate (1.70 g, 27 mmol) in anhydrousMeOH (12 mL) was added 10% Pd--C (400 mg) in one portion. After 2 h atroom temperature, the final solution was filtered over Celite,concentrated and the residue was directly subjected toflash-chromatography (CHCl₃ /MeOH 9:1) to provide 1.40 g (98%) of aslightly yellow oil: ¹ H NMR (300 MHz, CD₃ SOCD₃) δ1.40 (s, 9H), 5.22(s, 2H), 6.25-6.35 (m, 2H), 6.93 (br t, J=8.0 Hz, 1H), 8.29 (br s, 1H);¹³ C NMR (75 MHz, CDCl₃) δ28.5, 80.4, 102.1 (d, J_(CF) =24 Hz), 110.7,117.2, 122.8, 143.4, 153.1, 154.1 (d, J_(CF) =244 Hz); HRMS (EI) m/zcalcd for C₁₁ H₁₅ FN₂ O₂ (M⁺) 226.1118, found 226.1116; LRMS (EI) m/z226 (M⁺), 170, 126, 83, 57.

(3) 4-tert-Butyloxycarbonylamino-3-fluorophenyl isonitrile (25)

To a stirred solution of dicyclohexylcarbodiimide (1.51 g, 7.31 mmol) inCH₂ Cl₂ (15 mL) at 0° C. was added formic acid (275 mL, 7.31 mmol)dropwise. After 10 minutes, the resulting mixture was added over aperiod of 5 minutes to a solution of the compound prepared in Example(2) (1.28 g, 5.66 mmol) in CH₂ Cl₂ (10 mL) and pyridine (0.61 mL, 7.50mmol) at 0° C. The reaction mixture was then allowed to warm to roomtemperature and stirred 16 h. After filtration over Celite, the finalsolution was concentrated and subjected to flash-chromatography (CHCl₃/AcOEt 85:15) to give 1.44 g (100%) of4-tert-butyloxycarbonylamino-3-fluoroformamide, as a white solid. Thisintermediate (1.38 g, 5.43 mmol) was dissolved in CH₂ Cl₂ (20 mL) and,at 0° C., were successively added tetrabromomethane (1.93 g, 5.80 mmol),triphenylphosphine (1.52 g, 5.80 mmol), and1.4-diazabicyclo[2.2.2]octane (DABCO, 650 mg, 5.80 mmol). The reactionmixture was allowed to warm to room temperature and stirred 2 h. Afterevaporation of the solvent, the crude was triturated in ice-cooled Et₂ O(20 mL) and filtered over Celite. The residue obtained after evaporationof the solvent was purified by flash-chromatography (hexanes/AcOEt 95:5to 9:1) to provide 660 mg (51%) of a slightly brown solid: ¹ H NMR (300MHz, CDCl₃) δ1.51 (s, 9H), 6.76 (br s, 1H), 7.05-7.20 (m, 2H), 8.17 (brt, J=8.6 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ28.1, 81.8, 113.3 (d, J_(CF)=25 Hz), 119.7, 123.0, 128.6, 150.6 (d, J_(CF) =242 Hz), 151.8, 164.2;HRMS (EI) m/z calcd for C₁₂ H₁₃ FN₂ O₂ (M⁺) 236.0961, found 236.0952;LRMS (EI) m/z 236 (M⁺), 180, 163, 136, 08, 57.

(4)(20S)-10-tert-Butyloxycarbonylamino-11-fluoro-7-trimethylsilyl-camptothecin(26) and(20S)-10-tert-butyloxycarbonylamino-9-fluoro-7-trimethylsilylcamptothecin(27) (mixture respectively 1.9:1) ##STR21##

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (66.8 mg, 0.15 mmol) and the compounddescribed in Example (3) (110 mg, 0.50 mmol) provided, afterflash-chromatographies (CHCl₃ /MeOH 96:4; CHCl₃ /acetone 10:1), 47.6 mg(57%) of a slightly yellow oil containing the above regioisomers: ¹ HNMR (300 MHz, CDCl₃) δ0.54 (d, J=4.9 Hz, 9H_(minor)), 0.65 (s,9H_(major)), 0.99 (t, J=7.3 Hz, 3H), 1.86 (m, 2H), 3.93 (br s, 1H), 5.24(d, J=16.3 Hz, 1H_(minor)), 5.25 (br s, 2H_(major)), 5.25 (d, J=16.3 Hz,1H_(major)), 5.30 (br s, 2H_(minor)), 5.68 (d, J=16.3 Hz, 1H_(minor)),5.69 (d, J=16.3 Hz, 1H_(major)), 6.98 (d, J=3.6 Hz, 1H_(minor)), 7.02(d, J=3.6 Hz, 1H_(major)), 7.52 (s, 1H_(minor)), 7.53 (s, 1H_(major)),7.74 (d, J=12.1 Hz, 1H_(major)), 7.92 (br d, J=9.3 Hz, 1H_(minor)), 8.60(br t, J=8.4 Hz, 1H_(minor)), 9.08 (d, J=8.7 Hz, 1H_(major)); HRMS (EI)m/z calcd for C₂₈ H₃₂ FN₃ O₆ Si 553.2044, found 553.2022; LRMS (EI) m/z553 (M⁺), 493, 479, 453, 435, 424, 409, 394, 380, 353.

(5) (20S)-10-Amino-11-fluoro-7-trimethylsilylcamptothecin (28)

The compound prepared in Example (4) (41.3 mg, 0.0746 mmol) wasdeprotected following the conditions described in Example 9-(3). Afterworkup, the crude was subjected to a flash-chromatography (CHCl₃/acetone/MeOH 70:10:1.5) to provide, in order of elution, first 14.1 mg(42%) of the pure(20S)-10-amino-11-fluoro-7-trimethylsilyl-camptothecin, then a 15.2 mgof a c.a. 1:1 mixture of(20S)-10-amino-11-fluoro-7-trimethylsilylcamptothecin and(20S)-10-amino-9-fluoro-7-trimethylsilylcamptothecin. Thecharacteristics of (20S)-10-amino-11-fluoro-7-trimethylsilylcamptothecinare as follows: [α]_(D) ²⁰ +20.0 (c 0.2, CHCl₃ /MeOH 4:1); ¹ H NMR (300MHz, CDCl₃) δ0.59 (s, 9H), 1.00 (t, J=7.4 Hz, 3H), 1.86 (m, 2H), 3.86(br s, 1H), 4.31 (br s, 2H), 5.21 (br s, 2H), 5.26 (d, J=16.4 Hz, 1H),5.69 (d, J=16.4 Hz, 1H), 7.30 (d, J=9.3 Hz, 1H), 7.50 (s, 1H), 7.69 (d,J=11.8 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃ /CD₃ OD 10:1) δ1.4, 7.7, 31.4,51.9, 66.1, 72.7, 97.1, 109.4, 113.6 (d, J_(CF) =20 Hz), 117.3, 130.8,134.4, 136.4, 140.2, 142., 146.5, 147.6, 150.6, 153.9, 154.0 (d, J_(CF)=251 Hz), 157.6, 173.9; HRMS (EI) m/z calcd for C₂₃ H₂₄ FN₃ O₄ Si (M⁺)453.1520, found 453.1500; LRMS (EI) m/z 453 (M⁺), 424, 409, 394, 352,181, 131, 119.

EXAMPLE 11

Preparation of (20S)-11,12-difluoro-7-trimethylsilylcamptothecin and(20S)-9,10-difluoro-7-trimethylsilylcamptothecin (mixture respectively3:1) ##STR22##

Following the procedure described in Example 1-(2), the compoundprepared in Example 1-(1) (44.5 mg, 0.10 mmol) and 2,3-difluorophenylisonitrile [prepared in 20% yield following the procedure of Weber, W.P. et al., Tetrahedron Lett., 13, 1637 (1972) with stirring 2 days atroom temperature before workup] (42 mg, 0.30 mmol) afforded, afterflash-chromatographies (CHCl₃ /MeOH 95:5; CHCl₃ /acetone 10:1 to 4:1),22.6 mg (50%) of a slightly yellow oil containing the aboveregioisomers: ¹ H NMR (300 MHz, CDCl₃) δ0.56 (d, J=4.8 Hz, 1H_(minor)),0.65 (s, 9H_(major)), 1.00 (t, J=7.4 Hz, 3H), 1.86 (m, 2H), 3.87 (br s,1H_(minor)), 3.97 (br s, 1H_(major)), 5.0-5.47 (m, 3H), 5.68 (d, J=16.5Hz, 1H), 5.70 (d, J=16.4 Hz, 1H_(minor)), 7.31 (m, 1H_(minor)), 7.44(dt, J=9.4, 7.4 Hz, 1H_(major)), 7.59 (s, 1H_(minor)), 7.60 (s,1H_(major)), 7.68 (m, 1H_(minor)), 7.93 (m, 1H_(major)); HRMS (EI) m/zcalcd for C₂₃ H₂₂ F₂ N₂ O₄ Si (M⁺) 456.1317, found 456.1321; LRMS (EI)m/z 456 (M⁺), 438, 428, 412, 383, 356, 327.

EXAMPLE 12

Preparation of 20S-7-triisopropylsilylcamptothecin ##STR23## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(triisopropylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure outlined in example 1-(1), iodopyridone 2, (200mg, 0.598 mmol) was combined with triisopropylsilyl-2-propynyl bromide(329 mg, 1.196 mmol). Chromatography (CH₂ Cl₂ /AcOEt 9:1) gave 41.1 mg(13%) of a white foam: ¹ H NMR (300 MHz, CDCl₃) δ0.91 (t, J=7 Hz, 6H),0.99 (s, 18H), 1.71 (m, J=7 Hz, 2H), 3.65 (s, 1H), 5.0-5.2 (m, 3H), 5.45(d, J=16 Hz, 1H), 7.13 (s, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ7.7, 11.2,18.7, 31.7, 44.6, 66.5, 71.9, 87.7, 100.1, 116.6, 118.2, 148.6, 158.0,173.4; HRMS (EI) m/z calcd for C₂₂ H₃₂ INO₄ Si (M⁺) 529.1162, found529.1145; LRMS (EI) m/z 529 (M⁺), 486, 442, 82, 59.

(2) (20S)-7-Triisopropylsilylcamptothecin

Following the procedure outlined in example 1-(2), the pyridonedescribed above (41 mg, 0.077 mmol) yielded 23.3 mg (60%) of a lightyellow solid: [α]_(D) ²⁰ +31.7 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3026,3008, 2996, 2962, 2950, 2932, 2892, 2869, 1742, 1658, 1598, 1555, 1466,1230, 1220, 1158; ¹ H NMR (300 MHz, CDCl₃) δ1.02 (t, J=7 Hz, 3H), 1.18(d, J =7 Hz, 18H), 1.60-2.0 (m, 5H), 2.17 (s, 1H), 5.31 (d, J =16 Hz,1H), 5.41 (s, 2H), 5.76 (d, J=16, 1H), 7.61 (t, J=7 Hz, 1H), 7.69 (s,1H), 7.78 (t, J=7 Hz 1H), 8.20 (t, J=7 Hz, 2H); ¹³ C NMR (125 MHz,CDCl₃) δ7.9, 13.5, 19.2, 31.7, 52.6, 66.5, 72.9, 98.4, 118.6, 127.1,129.7, 130.2, 130.4, 133.6, 136.3, 145.0, 146.0, 150.3, 150.6, 157.4,174.1; HRMS (EI) m/z calcd for C₂₉ H₃₆ N₂ O₄ Si (M⁺) 504.2444, found504.2436; LRMS (EI) m/z 504 (M⁺), 461, 433, 419, 405, 391, 375, 361,347, 311, 275, 174, 93, 69, 59.

EXAMPLE 13

Preparation of 20S-7-triisopropylsilylcamptothecin ##STR24## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(triethylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure outlined in example 1-(1), iodopyridone 2, (150mg, 0.450 mmol) was combined with triethylsilyl-2-propynyl bromide (210mg, 0.90 mmol). Chromatography (CH₂ Cl₂ /AcOEt 9:1) gave 97.0 mg (45%)of a white foam: ¹ H NMR (300 MHz, CDCl₃) δ0.54 (q, J=8 Hz, 6H), 0.92(t, J=8 Hz, 12H), 1.74 (m, J=7 Hz, 2H), 3.57 (s, 1H), 4.9-5.1 (m, 3H),5.46 (d, J=16 Hz, 1H), 7.13 (s, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ4.1, 7.4,7.6, 31.5, 44.5, 66.3, 71.8, 88.7, 99.2, 100.0, 116.5, 118.1, 148.5,158.0, 173.2; HRMS (EI) m/z calcd for C₁₉ H₂₆ INO₄ Si (M⁺) 487.0676,found 487.0688; LRMS (EI) m/z 487 (M⁺), 458, 430, 420, 402, 360, 332,153, 141, 125, 96, 83, 68, 57.

(2) (20S)-7-Triethylsilylcamptothecin

Following the procedure outlined in example 1-(2), the pyridonedescribed above (48.7 mg, 0.1 mmol) yielded 29.8 mg (65%) of a lightyellow solid: [α]_(D) ²⁰ +35.9 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3015,3002, 2960, 2935, 1741, 1658, 1599, 1219, 1199, 1158; ¹ H NMR (300 MHz,CDCl₃) δ0.80-1.00 (m, 12H), 1.0-1.18 (m, 6H), 1.70-1.90 (m, 2H),5.22-5.27 (m, 3H), 5.69 (d, J=16 Hz, 1H), 7.58 (t, J=7 Hz, 1H), 7.63 (s,1H), 7.72 (t, J=7 Hz 1H), 8.18 (m, 2H); ¹³ C NMR (125 MHz, CDCl₃) δ5.0,7.6, 7.9, 31.7, 52.1, 66.5, 72.9, 97.7, 118.3, 127.4, 127.9, 129.7,131.2, 132.6, 136.1, 142.6, 146.6, 147.9, 150.2, 150.9, 157.6, 174.1;HRMS (EI) m/z calcd for C₂₆ H₃₀ N₂ O₄ Si (M⁺) 462.1975, found 462.1982;LRMS (EI) m/z 462 (M⁺), 433, 418, 405, 389, 361, 256, 220, 205, 189,178, 149, 137, 123, 109, 95, 81, 69, 57.

EXAMPLE 14

Preparation of (20S)-7-(dimethyl-(1'S,2'S,5'S) 7,7dimethylnorpinylsilyl)camptothecin ##STR25## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(dimethyl-(1S,2S,5S) 7,7dimethylnorpinylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure outlined in example 1-(1), iodopyridone 2 (150mg, 0.450 mmol) was combined with dimethyl-(1S, 2S, 5S)7,7dimethylnorpinylsilyl-2-propynyl bromide (281 mg, 0.90 mmol).Chromatography (CH₂ Cl₂ /AcOEt 9:1) gave 100.8 mg (39%) of a white foam:¹ H NMR (300 MHz, CDCl₃) δ0.10 (d, J=2 Hz, 6H), 0.48-0.70 (m, 2H), 0.72(s, 3H), 0.93 (t, J=7 Hz, 3H), 1.10 (s, 3H), 1.15-1.40 (m, 3H),1.60-1.85 (m, 6H), 1.88-2.00 (m, 1H), 2.05-2.20 (m, 1H), 3.58 (s, 1H),4.95 (m, 3H), 5.46 (d, J=16 Hz, 1H), 7.13 (s, 1H); ¹³ C NMR (75 MHz,CDCl₃) δ0.78, 7.8, 20.2, 23.1, 24.0, 24.8, 25.3, 27.0, 31.3, 31.7, 39.7,40.7, 44.7, 49.1, 66.5, 71.9, 91.0, 98.5, 100.3, 116.6, 118.3, 148.7,158.0, 173.4.

(2) (20S)-7-(dimethyl-(1'S,2'S,5'S) 7,7dimethylnorpinylsilyl)camptothecin

Following the procedure outlined in example 1-(2), the pyridonedescribed above (57.0 mg, 0.1 mmol) yielded 29.4 mg (54%) of a lightyellow solid: [α]²⁰ _(D) +29.2 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3020,3000, 2980, 2972, 2939, 2914, 2824, 2867, 1741, 1658, 1599, 1556, 1264,1231, 1201, 1157, 843; ¹ H NMR (300 MHz, CDCl₃) δ0.50-0.70 (m, 8H),0.90-1.10 (m, 9H), 1.10-1.35 (m, 4H), 1.40-1.60 (m, 3H), 1.72 (m, 1H),1.80-1.95 (m, 2H), 2.05-2.11 (m, 2H), 5.25 (d, J=16 Hz 1H), 5.27 (s,2H), 5.69 (d, J=16 Hz, 1H), 7.58 (t, J=8 Hz, 1H), 7.62 (s, 1H), 7.72 (t,J=8 Hz, 1H), 8.10-8.2 (m, 2H); ¹³ C NMR (125 MHz, CDCl₃) δ1.4, 7.9,19.9, 23.0, 24.6, 25.3, 26.8, 31.6; 31.7, 39.6, 40.5, 49.3, 52.0, 66.5,72.9, 97.7, 118.3, 127.3, 128.3, 129.7, 131.2, 132.1, 134.6, 144.6,146.6, 148.0, 150.2, 150.9, 157.6, 174.0; HRMS (EI) m/z calcd for C₃₂H₃₈ N₂ O₄ Si (M⁺) 542.2601, found 542.2588; LRMS (EI) m/z 542 (M⁺), 498,487, 460, 443, 431, 406, 387, 377, 362, 333, 318, 304, 289, 275, 219,178, 166, 141, 115, 95, 67.

EXAMPLE 15

(20S)-7-(3-cyanopropyldimethylsilyl)camptothecin ##STR26## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(3-cyanopropyldimethylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure cited by Rico and co-workers (J. Org. Chem.1994, 59, 415), iodopyridone 2, (150 mg, 0.450 mmol) was combined with3-cyanopropyldimethylsilyl-2-propynyl bromide (165 mg, 0.678 mmol), K₂CO₃ (124 mg, 0.90 mmol), Bu₄ N⁺ Br⁻ (14.5 mg, 0.045 mmol), H₂ O (0.02mL) and toluene (3.6 mL). This mixture was refluxed for 1 h. Afterfiltration and chromatography (CH₂ Cl₂ /AcOEt 9:1) 34.0 mg (15%) of awhite oil was obtained: ¹ H NMR (300 MHz, CDCl₃) δ0.17 (s, 6H),0.70-0.80 (m, 2H), 0.98 (t, J=7 Hz, 3H), 1.70-1.90 (m, 4H), 2.39 (t,J=7, 2H), 3.66 (s, 1H), 4.9-5.22 (m, 3H), 5.51 (d, J=16 Hz, 1H), 7.19(s, 1H); ¹³ C NMR (125 MHz, CDCl₃) δ-2.1, 7.8, 15.4, 20.5, 20.6, 31.6,44.6, 66.4, 71.9, 89.1, 99.6, 100.0, 116.7, 118.3, 119.7, 148.8, 158.0,173.3; HRMS (EI) m/z calcd for C₁₉ H₂₃ IN₂ O₄ Si (M⁺) 498.0472, found498.0480; LRMS (EI) m/z 498 (M⁺), 483, 470, 445, 430, 416, 402, 392,371, 348, 335, 306, 290, 266, 223, 202, 185, 163, 136, 126, 109, 98, 81,69, 57.

(2) (20S)-7-(3-cyanopropyldimethylsilyl)camptothecin

Following the procedure outlined in example 1-(2), the pyridonedescribed above (25.0 mg, 0.05 mmol) yielded 9.8 mg (41%) of a lightyellow solid: [α]_(D) ²⁰ +34.3 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3025,3016, 1741, 1659, 1600, 1264, 1222; ¹ H NMR (300 MHz, CDCl₃) δ0.71 (s,6H), 1.05 (t, J=7 Hz, 3H), 1.26 (m, 2H), 1.66 (m, 2H), 1.90 (m, 2H),2.35 (t, J=7 Hz, 2H), 3.76 (s, 1H), 5.31 (d, J=16 Hz, 1H), 5.31 (s, 2H),5.75 (d, J=16 Hz, 1H), 7.67 (m, 2H), 7.82 (t, J=8 Hz, 1H), 8.17 (d, J=8Hz 1H), 8.24 (d, J=8 Hz, 1H); ¹³ C NMR (125 MHz, CDCl₃) δ0.2, 7.9, 16.8,20.7, 20.73, 31.7, 50.9, 66.5, 72.8, 97.9, 118.5, 119.2, 127.7, 127.8,130.0, 131.4, 131.9, 135.2, 141.9, 146.3, 148.1, 150.3, 151.1, 157.5,174.0; HRMS (EI) m/z calcd for C₂₆ H₂₇ N₃ O₄ Si (M⁺) 473.1771, found473.1755; LRMS (EI) m/z 473 (M⁺), 444, 429, 414, 400, 389, 373 362, 344,331, 303, 289, 2.75, 245, 219, 166, 152, 130, 98, 71.

EXAMPLE 16

Preparation of (20S)-7-(3-halopropyldimethylsilyl) camptothecin (thechloropropyl derivative is DB-148) ##STR27## (1)(S)-4-Ethyl-4-hydroxy-6-iodo(and6-bromo)-3-oxo-7-(3-chloropropyldimethylsilyl-2-propynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure outlined in example 1-(1), [iodopyridone 2 (150mg, 0.450 mmol) was combined with 3-chloropropyldimethylsilyl-2-propynylbromide (228 mg, 0.90 mmol) Chromatography (CH₂ Cl₂ /AcOEt 9:1) gave75.4 mg (33%) of a clear oil. Analysis of the NMR showed the presence ofthe alkyl bromide in addition to the desired chloro derivative in a1.6:1 ratio in favor of the former.: ¹ H NMR (300 MHz, CDCl₃) δ0.09 (s,6H), 0.60-0.70 (m, 2H), 0.85-0.89 (t, J=7 Hz, 3H), 1.60-1.95 (m, 4H),3.33 (t, J=7 Hz, 2H, assigned to iodo), 3.44 (t, J=7 Hz, 2H, assigned tobromo), 3.75 (s, 1H), 4.91-5.18 (m, 3H), 5.42 (d, J=16 Hz, 1H), 7.12 (s,1H).

(2) (20S)-7-(3-halopropyldimethylsilyl)camptothecin

Following the procedure outlined in example 1-(2), the pyridonedescribed above (51 mg, 0.1 mmol) yielded 23 mg (49%) of a light yellowsolid. Analysis of the spectral data identified this solid as a 3component mixture corresponding to the chloro, bromo and the iododerivatives in a 1.6:1:1.3 ratio: [α]_(D) ²⁰ +30.8 (c 0.2, CH₂ Cl₂); IR(CHCl₃, cm⁻¹) 3029, 3012, 2980, 2963, 2933, 1742, 1658, 1600, 1556,1258, 1233, 1218, 1200, 1158, 1045, 843, 822, 794; ¹ H NMR (300 MHz,CDCl₃) δ0.69 (s, 6H), 1.04 (t, J=7 Hz, 3H), 1.18-1.30 (m, 2H), 1.60-2.0(m, 4H), 3.15 (t, J=7 Hz, 2H, assigned to iodo), 3.36 (t, J=7 Hz, 2H,assigned to bromo), 3.48 (t, J=7 Hz, 2H, assigned to chloro), 3.88 (s,1H), 5.30 (d, J=16 Hz, 1H), 5.31 (s, 2H), 5.74 (d, J=16 Hz, 1H),7.62-7.66 (m, 2H), 7.87 (t, J=8 Hz, 1H), 8.18 (d, J =8 Hz, 1H), 8.22 (d,J=8 Hz, 1H); ¹³ C NMR (125 MHz, CDCl₃) δ0.2, 7.9, 14.7, 27.5, 31.7,47.4, 51.9, 66.4, 72.8, 98.2, 118.6, 127.7, 127.9, 130.0, 131.0, 132.0,135.2, 146.1, 147.6, 150.2, 157.5, 174.0; HRMS (EI) m/z calcd for C₂₅H₂₇ ClN₂ O₄ Si (M⁺) 482.1429, found 482.1413; LRMS (EI) m/z 482 (M⁺),453, 438, 361, 305, 275.

EXAMPLE 17

Preparation of (20S)10-acetoxy-7-tert-butyldimethylsilylcamptothecin##STR28##

Following the procedure outlined in example 1-(2), the pyridonedescribed above (34.5 mg, 0.071 mmol) and p-acetoxyisonitrile yielded21.3 mg (58%) of a light yellow solid: [α]_(D) ²⁰ +36.2 (c 0.2, CH₂Cl₂); IR (CHCl₃, cm⁻¹) 3029, 3000, 2958, 2931, 2902, 2885, 2859, 1742,1659, 1600, 1557, 1504, 1464, 1371, 1256, 1232, 1195, 1166, 1045; ¹ HNMR (300 MHz, CDCl₃) δ0.69 (s, 6H), 0.90 (s, 9H), 1.04 (t, J=7 Hz, 3H),1.80-2.00 (m, J=7 Hz, 2H), 2.40 (s, 3H), 3.81 (s, 1H), 5.30 (d, J=16 Hz1H), 5.31 (s, 2H), 5.75 (d, J =16 Hz, 1H), 7.53 (dd, J₁ =9 Hz, J₂ =2 Hz,1H), 7.65 (s, 1H), 8.08 (d, J=2 Hz, 1H), 8.21 (d, J=9 Hz, 1H); ¹³ C NMR(125 MHz, CDCl₃) δ0.6, 7.9, 19.3, 21.5, 27.2, 31.7, 52.5, 66.5, 72.9,97.7, 118.4, 120.4, 124.8, 132.1, 133.2, 136.7, 142.8, 146.2, 146.4,149.0, 150.2, 150.8, 157.5, 169.1, 174.1; LRMS (EI) m/z 520 (M⁺), 478,463, 421, 377, 347, 320, 291, 57.

EXAMPLE 18

(2) (20S)10-Acetoxy-7-tert-butyldimethylsilylcamptothecin

Following the procedure outlined in example 2-(2), the pyridonedescribed above (34.5 mg, 0.071 mmol) yielded, using the samechromatographic conditions, 21.3 mg (58%) of a light yellow solid:[α]_(D) ²⁰ +36.2 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3029, 3000, 2958,2931, 2902, 2885, 2859, 1742, 1659, 1600, 1557, 1504, 1464, 1371, 1256,1232, 1195, 1166, 1045; ¹ H NMR (300 MHz, CDCl₃) δ0.69 (s, 6H), 0.90 (s,9H), 1.04 (t, J=7 Hz, 3H), 1.80-2.00 (m, J=7 Hz, 2H), 2.40 (s, 3H), 3.81(s, 1H), 5.30 (d, J=16 Hz 1H), 5.31.(s, 2H), 5.75 (d, J=16 Hz, 1H), 7.53(dd, J₁ =9 Hz, J₂ =2 Hz, 1H), 7.65 (s, 1H), 8.08 (d, J=2 Hz, 1H), 8.21(d, J=9 Hz, 1H); ¹³ C NMR (125 MHz, CDCl₃) δ0.6, 7.9, 19.3, 21.5, 27.2,31.7, 52.5, 66.5, 72.9, 97.7, 118.4, 120.4, 124.8, 132.1, 133.2, 136.7,142.8, 146.2, 146.4, 149.0, 150.2, 150.8, 157.5, 169.1, 174.1; HRMS (EI)m/z calcd for C₂₈ H₃₂ N₂ O₆ Si (M⁺) 520.2030, found 520.2014 LRMS (EI)m/z 520 (M⁺), 478, 463, 421, 377, 347, 320, 291, 57.

EXAMPLE 19 ##STR29##(20S)10-Hydroxy-7-tert-butyldimethylsilylcamptothecin (DB-67)

Following the procedure outlined in example 5, (13.4 mg, 0.026 mmol) ofthe compound described in example 18 was converted to the hydroxyderivative. Purification (2:1 CH₂ Cl_(2:) Acetone) on a preparative TLCplate gave 10.6 mg (85%) of a yellow solid: [α]_(D) ²⁰ +17.4 (c 0.2, 3:1CH₂ Cl₂ /MeOH); ¹ H NMR (300 MHz, 3:1 CDCl₃ /CD₃ OD) δ0.66 (s, 6H),0.88-1.05 (m, 12H), 1.80-2.00 (m, 2H), 5.25-5.30 (m, 3H), 5.70 (d, J=16Hz, 1H), 7.37 (dd, J₁ =9 Hz, J₂ =2 Hz, 1H), 7.54 (d, J=2 Hz, 1H), 7.60(s, 1H), 8.05 (d, J=9 Hz, 1H); ¹³ C NMR (125 MHz, (3:1) CDCl_(3:) CD₃OD) δ8.1, 20.6, 27.6, 30.4, 31.9, 53.6, 66.5, 73.9, 98.6, 112.1, 118.8,123.3, 132.1, 135.6, 137.4, 141.6, 143.8, 147.3, 148.4, 152.6, 157.5,158.7, 174.7; HRMS (EI) m/z calcd for C₂₆ H₃₀ N₂ O₅ Si (M⁺) 478.1924,found 478.1947 LRMS (EI) m/z 478 (M⁺), 434, 421, 377, 304, 284, 227,178, 149, 137, 109, 97, 83, 69, 57.

EXAMPLE 20

Preparation of (20S)-7-(trimethylsilylmethyl)camptothecin ##STR30## (1)(S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(4-trimethylsilyl-2-butynyl)-1H-pyrano[3,4-c]-8-pyridone

Following the procedure described in Example 1-(1), iodopyridone (2)(200 mg, 0.60 mmol) and 4-trimethylsilyl-2-butynyl bromide (245 mg, 1.20mmol) gave, after flash-chromatography (CH₂ Cl₂ /AcOEt 9:1), 77.7 mg(28%) of a white foam: [α]_(D) ²⁰ +62.7 (c 0.2, CHCl₃); IR (CHCl₃, cm⁻¹)3540, 3026, 2955, 1742, 1649, 1607, 1529, 1250, 1219, 1208, 1158, 1140;¹ H NMR (300 MHz, CDCl₃) δ0.06 (s, 9H), 0.92 (t, J=7.4 Hz, 3H), 1.44 (t,J=2.4 Hz, 2H), 1.76 (m, J=7.4 Hz, 2H), 3.74 (s, 1H), 4.98 (br s, 2H),5.07 (d, J=15 Hz, 1H), 5.48 (d, J=16.4 Hz, 1H), 7.15 (s, 1H); ¹³ C NMR(75 MHz, CDCl₃) δ-1.96, 7.5, 7.6, 31.5, 44.8, 66.3, 71.7, 84.3, 100.3,116.3, 118.1, 148.3, 157.9, 173.3; HRMS (EI) m/z calcd for C₁₇ H₂₂ INO₄Si (M⁺) 459.0352, found 459.0363; LRMS (EI) m/z 459 (M⁺), 444, 386, 348,73, 57.

(2) (20S)-7-(Trimethylsilylmethyl)camptothecin

Following the procedure described in Example 1-(2), the compoundprepared in (1) (46 mg, 0.10 mmol) yielded, after flash chromatographies(CH₂ Cl₂ /MeOH 96:4; CH₂ Cl₂ /acetone 9:1), 16.2 mg (38%) of a lightyellow solid: [α]_(D) ²⁰ +37.6 (c 0.2, CHCl₃); IR (CHCl₃, cm⁻¹) 3002,2984, 2962, 1741, 1659, 1601, 1572, 1559, 1253, 1219, 1197, 1157, 849; ¹H NMR (500 MHz, CDCl₃) δ-0.34 (s, 9H), 0.62 (t, J=7.3 Hz, 3H), 1.48 (m,2H), 2.31 (d, J=3.0 Hz, 2H), 3.53 (s, 1H), 4.74 (s, 2H), 4.89 (d, J=16.2Hz, 1H), 5.34 (d, J=16.2 Hz, 1H), 6.85 (s, 1H), 7.20 (t, J=7.8 Hz, 1H),7.36 (t, J=7.4 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.78 (d, J=8.4 Hz, 1H);¹³ C NMR (75 MHz, CDCl₃) δ-0.32, 1.1, 7.9, 31.6, 50.3, 66.4, 72.9, 98.1,112.3, 124.4, 125.84, 126.91, 127.2, 130.1, 130.5, 144.6, 147.4, 149.2,150.3, 151.5, 157.7, 174.1; HRMS (EI) m/z calcd for C₂₄ H₂₆ N₂ O₄ Si(M⁺) 434.1676, found 434.1662; LRMS (EI) m/z 434 (M⁺), 390, 362, 316,290, 242, 223, 185, 147, 93, 73.

EXAMPLE 21

(20S)-10-Hydroxy-7-[(2-trimethylsilyl)ethyl]camptothecin (36a) (DB-174)##STR31## (1) (4,4-Dibromo-but-3-enyl)trimethylsilane

This known compound was prepared by a modification of the procedure inPiers, E.; Gavai, A. V. J. Org. Chem. 1990, 55, 2374. To a flame driedflask was added dry CH₂ Cl₂ (150 mL) and triphenylphosphine (16 g, 61.2mmol). The temperature was lowered to 0° C. and CBr₄ (10.15 g, 30.6mmol) was added portionwise. After 30 min, a solution of3-trimethylsilylpropanal (2.0 g, 15.3 mmol) in dry CH₂ Cl₂ (20 ml) wasadded. After 1 h at 0° C., the reaction was diluted with ether andfiltered through celite. The celite was rinsed with ether (4×50 mL) andthe combined ether solution was extracted with H₂ O (100 mL), saturatedNaHCO₃ (100 mL), saturated NH₄ Cl (100 mL) and brine (100 mL). Theorganic layer was dried (MgSO₄), filtered and evaporated to give a crudewhite solid. The solid residue was washed with pentane. The pentanesolution was concentrated and the oily residue was chromatographed onsilica gel (pentane 100%) to give(4,4-dibromo-but-3-enyl)trimethylsilane as a clear oil weighing 3.7 g. ¹H NMR showed 27% contamination by remaining pentane which gives acorrected yield of 62%: IR (neat, cm⁻¹) 2953, 2922, 2898, 1620, 1443,1413, 1249, 1176, 1034, 986, 859, 837; ¹ H NMR (300 MHz, CDCl₃) δ0.03(s, 9H), 0.62-0.67 (m, 2H), 2.06-2.14 (m, 2H), 6.41 (t, J=7 Hz 1H); ¹³ CNMR (75 MHz, CDCl₃) δ-1.9, 15.1, 27.7, 87.5, 141.2.

(2) 5-Trimethylsilanylpent-2-yn-1-ol (40)

To a flame dried flask was added (4,4-dibromo-but-3-enyl)trimethylsilane(3.43 g, 12 mmol). Dry THF (150 mL) was added and the mixture was cooledto -78° C. BuLi 1.6N in hexanes (24 mmol, 15 mL) was added and themixture was stirred 1 h at -78° C., warmed to 22° C., and stirred for anadditional 1 h. Finally, paraformaldehyde (1.4 g) was added and themixture was refluxed. After 3.5 h the reaction was cooled to 22° C. andsat. NH₄ Cl (50 mL) was added. The contents of the flask weretransferred to a separatory funnel and extracted with ether (3×200 mL).The organic layer was dried (Na₂ SO₄), filtered and evaporated to give acrude yellow oil. Flash chromatography on silica gel (pentane/ether 5:1)afforded 1.57 g (84%) of 5-trimethylsilanylpent-2-yn-1-ol: IR (neat,cm⁻¹) 3350, 2953, 2219, 1436, 1412, 1318, 1249, 1178, 1124, 1015, 905,853; ¹ H NMR (300 MHz, CDCl₃) δ-0.003 (s, 9H), 0.76 (t, J=8 Hz 2H), 1.75(s, 1H), 2.20-2.24 (m, 2H), 4.21 (br s, 2H); ¹³ C NMR (75 MHz, CDCl₃)δ-1.3, 13.8, 16.4, 51.7, 78.2, 88.9; HRMS (EI) m/z calcd for C₇ H₁₃ OSi(M-CH₃) 141.0736, found 141.0733 LRMS (EI) m/z 141 (M-CH₃), 123, 113,103, 97, 91, 85, 75, 66, 59.

(3) (5-Bromopent-3-ynyl)trimethylsilane (41)

To a flame dried flask was added PPh₃ (1.76 g, 6.73 mmol) followed bydry CH₂ Cl₂ (60 mL). The mixture was placed in an ice bath and bromine(0.34 mL, 6.41 mmol) was added dropwise. A small amount of PPh₃ wasadded until the reaction went from yellow to clear in color. After 0.5 hat 0° C., 5-trimethylsilanylpent-2-yn-1-ol (1.0 g, 6.41 mmol) wasdissolved in CH₂ Cl₂ (5 mL) and added dropwise. After 4 h at 0° C., thereaction mixture was poured into a separatory funnel, diluted withpentane (250 mL) and extracted with H₂ O (100 mL) and sat. NaHCO₃ (100mL). The organic layer was dried (MgSO₄), filtered and reduced in volumeto 50 mL. The crude solution was chromatographed on a pad of silica gelwith pentane (500 mL). After evaporation of the pentane,(5-bromopent-3-ynyl)trimethylsilane (1.2 g 87%) was obtained as a clearoil: IR (neat, cm⁻¹) 2953, 2922, 2899, 2230, 1431, 1317, 1248, 1208,852; ¹ H NMR (300 MHz, CDCl₃) δ0.03 (s, 9H), 0.79 (t, J=8 Hz, 2H), 2.28(tt, J₁ =8 Hz, J₂ =2 Hz, 2H), 3.92 (t, J=2 Hz, 2H); ¹³ C NMR (75 MHz,CDCl₃) δ1.7, 13.6, 15.7, 15.8, 74.6, 90.2; HRMS (EI) m/z calcd for C₇H₁₂ BrSi (M-CH₃) 202.9892, found 202.9889 LRMS (EI) m/z 203 (M-CH₃),137, 73, 66.

(4)(20S)-4-Ethyl-4-hydroxy-6-iodo-3-oxo-7-(5-trimethylsilanylpent-2-ynyl)-1H-pyrano[3,4-c]-8-pyridone(43)

Following the procedure described in Example 1-(1), iodopyridone (2),(0.2 g, 0.6 mmol)] and (5-bromopent-3-ynyl)trimethylsilane (260 mg, 1.19mmol) provided after flash chromatography (CH₂ Cl₂ /EtOAc 95:5) 0.21 g(74%) of(20S)-4-ethyl-4-hydroxy-6-iodo-3-oxo-7-(5-trimethylsilanyl-pent-2-ynyl)-1H-pyrano[3,4-c]-8-pyridoneas a white foam: [α]_(D) ²⁰ +54.4 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹)2952, 1746, 1648, 1528, 1427, 1138, 856, 755; ¹ H NMR (300 MHz, CDCl₃)δ-0.03 (s, 9H), 0.76 (t, J=8 Hz, 2H), 0.96 (t, J=7 Hz, 3H), 1.70-2.00(m, J=7 Hz, 2H), 2.16 (t, J=8 Hz, 2H), 3.77 (s, 1H), 5.04 (s, 2H), 5.10.(d, J=16 Hz, 1H), 5.49 (d, J=16 Hz, 1H), 7.16 (s, 1H); ¹³ C NMR (75 MHz,CDCl₃) δ-1.6, 7.8, 13.6, 15.7, 31.7, 44.8, 66.5, 71.9, 72.4, 88.1,100.5, 116.6, 118.3, 148.6, 158.2, 173.5; HRMS (EI) m/z calcd for C₁₈H₂₄ INO₄ Si (M⁺) 473.0519, found 473.0507 LRMS (EI) m/z 473 (M⁺), 458,386, 360, 346, 139, 73, 57.

(5) (20S)-10-Acetoxy-7-[(2-trimethylsilyl)ethyl]camptothecin (45)

Following the procedure described in Example 1-(2), a mixture of theiodopyridone prepared in (4) above (56.8 mg, 0.12 mmol) andp-acetoxyphenyl isonitrile (48 mg, 0.3 mmol) provided after flashchromatography (CH₂ Cl₂ /Acetone 10:1) 33.2 mg (55%) of(20S)-10-acetoxy-7-[(2-trimethylsilyl)ethyl]camptothecin as a tan solid:[α]_(D) ²⁰ +21.0 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 3039, 2996, 2954,1744, 1660, 1602, 1509, 1371, 1229, 1177; ¹ H NMR (300 MHz, CDCl₃) δ0.17(s, 9H), 0.88-0.95 (m, 2H), 1.03 (t, J=7 Hz, 3H), 1.80-2.00 (m, J=7 Hz,2H), 2.42 (s, 3H), 3.00 (m, 2H), 4.01 (br s, 1H), 5.22 (s, 2H), 5.30 (d,J=16 Hz, 1H), 5.74 (d, J=16 Hz, 1H), 7.54 (dd, J₁ =9 Hz, J₂ =2 Hz 1H),7.66 (s, 1H), 7.72 (d, J=2 Hz, 1H), 8.22 (d, J=2 Hz, 1H); ¹³ C NMR (125MHz, CDCl₃) δ-1.8, 7.9, 17.7, 21.4, 24.3, 31.7, 49.3, 66.4, 72.9, 98.1,114.5, 118.6, 125.4, 126.6, 127.2, 132.2, 146.8, 147.0, 147.5, 149.6,150.3, 151.9, 157.7, 169.4, 174.0; HRMS (EI) m/z calcd for C₂₇ H₃₀ N₂ O₆Si (M⁺) 506.1873, found 506.1869 LRMS (EI) m/z 506 (M⁺), 464, 436, 420,347, 336, 277, 193, 151, 109, 73.

(6) (20S)-10-Hydroxy-7-[(2-trimethylsilyl)ethyl]camptothecin (36a)

A mixture of the compound prepared in (5) above (17.7 mg, 0.035 mmol)and K₂ CO₃ (9.7 mg, 0.07 mmol) in MeOH (0.2 mL) and H₂ O (0.2 mL) wasstirred for 1.5 h at room temperature. The mixture was acidified withAcOH (8 drops), diluted with brine (10 mL) and extracted with EtOAc(10×20 mL). The combined organic layers were dried (Na₂ SO₄) andevaporated. The crude residue was subjected to two chromatographies (CH₂Cl_(2/) MeOH/AcOH 96:3:1 followed by CH₂ Cl₂ /Acetone 5:1), which gave7.6 mg (47%) of (20S)-10-hydroxy-7-[(2-trimethylsilyl)ethyl]camptothecinas a yellow solid: [α]_(D) ²⁰ +31.3 (c 0.2, CH₂ Cl₂ /MeOH 3:1); ¹ H NMR(300 MHz, CDCl₃) δ0.15 (s, 9H), 0.84-0.95 (m, 2H), 0.99 (t, J =7 Hz,3H), 1.80-2.00 (m, J=7 Hz, 2H), 2.99-3.05 (m, 2H), 5.20 (s, 2H), 5.29(d, J=16 Hz 1H), 5.62. (d, J=16 Hz 1H), 7.33 (d, J=2 Hz, 1H), 7.40 (dd,J₁ =9 Hz, J₂ =2 Hz, 1H), 7.63 (s, 1H), 8.01 (d, J=9 Hz, 1H); ¹³ C NMR(125 MHz, CDCl₃) δ-1.8, 8.3, 17.9, 25.1, 32.2, 66.9, 74.4, 99.1, 106.0,116.1, 119.7, 124.0, 128.4, 129.8, 132.3, 145.5, 146.8, 148.3, 150.1,153.0, 158.6, 159.4, 175.1; HRMS (EI) m/z calcd for C₂₅ H₂₈ N₂ O₅ Si(M⁺) 464.1767, found 464.1788 LRMS (EI) m/z 464 (M⁺), 420, 405, 391,364, 347, 167, 149, 104, 91, 73.

This compound showed activity inhibiting cell proliferation in severallines of glioma cells (U87, A172, SG388, T98G, LN-Z308) with medianeffective concentrations of 10-100 ng/ml.

EXAMPLE 22

(20S)-10-Amino-7-[2-trimethylsilyl)ethyl]camptothecin (36b) (DB-173)##STR32## (1)(20S)-10-tert-Butyloxycarbonylamino-7-[(2-trimethylsilyl)ethyl]camptothecin(45b)

Following the procedure described in Example 1-(2), a solution of theiodopyridone prepared in Example 21-(4) above (56.8 mg, 0.12 mmol) wasreacted with 4-tert-butyloxycarbonylaminophenyl isonitrile (65.4 mg, 0.3mmol). Column chromatography (CH₂ Cl₂ /Acetone 10:1) gave 38 mg (56%)(20S)-10-tert-butyloxycarbonylamino-7-[(2-trimethylsilyl)ethyl]camptothecinas a tan solid: [α]_(D) ²⁰ +18.5 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹)3019, 1738, 1658, 1600, 1531, 1215, 1155, 761; ¹ H NMR (300 MHz, CDCl₃)δ0.19 (s, 3H), 0.90-0.96 (m, 2H), 1.03 (t, J=7 Hz, 3H), 1.58 (s, 9H),1.8-2.0 (m, 2H), 3.02-3.08 (m, 2H), 3.89 (s, 1H), 5.21. (s, 2H), 5.30(d, J=16 Hz, 1H), 5.75 (d, J=16 Hz, 1H), 6.85 (br s, 1H), 7.57 (dd, J₁=9 Hz, J₂ =2 Hz, 1H), 7.61 (s, 1H), 8.11 (d, J=9 Hz, 1H), 8.31 (br s,1H); ¹³ C NMR (75 MHz, CDCl₃) δ-1.7, 8.0, 17.5, 24.4, 28.5, 31.8, 49.5,66.5, 73.0, 77.4, 81.4, 97.7, 110.1, 118.2, 123.3, 126.7, 127.6, 131.4,137.8, 146.2, 147.4, 150.37, 150.4, 152.6, 157.8, 174.1; HRMS (EI) m/zcalcd for C₃₀ H₃₇ N₃ O₆ Si (M⁺) 563.2452, found 563.2426 LRMS (EI) m/z463 (M-C₅ H₈ O₂), 419, 404, 363, 363, 346, 332, 289, 246, 149, 131, 73,57.

(2) (20S)-10-Amino-7-[2-trimethylsilyl)ethyl]camptothecin (36b)

The camptothecin derivative prepared in (2) above (17.5 mg, 0.031 mmol)was dissolved in CH₂ Cl₂ (1 mL) and trifluoroacetic acid (0.25 mL) wasadded. After 3 h at 22° C., the mixture was poured into saturated NaHCO₃(20 mL) and extracted with EtOAc (10×15 mL). The organic phase was dried(Na₂ SO₄), concentrated and chromatographed (CH₂ Cl₂ /Acetone 85:15), togive 9.4 mg (65%) of a yellow solid: [α]_(D) ²⁰ +17.0 (c 0.2, CH₂ Cl₂/MeOH 3:1); ¹ H NMR (300 MHz, CDCl₃) δ0.15 (s, 9H), 0.85-0.91 (m, 2H),0.99 (t, J =7 Hz, 3H), 1.87-2.05 (m, 2H), 2.85-2.98 (m, 2H), 5.04 (d,J=19 Hz, 1H), 5.09 (d, J=19 Hz 1H), 5.29. (d, J=16 Hz, 1H), 5.58 (d,J=16 Hz, 1H), 7.01 (d, J=2 Hz, 1), 7.25 (dd, J₁ =9 Hz, J_(s2) =2 Hz,1H), 7.54 (s, 1H), 7.84 (d, J=9 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃) δ1.8,8.1, 17.4, 24.5, 31.8, 50.0, 66.4, 73.8, 98.4, 102.3, 118.2, 123.4,127.2, 129.6, 131.3, 144.1, 145.2, 147.6, 147.9, 148.5, 152.4, 158.7,174.6; HRMS (EI) m/z calcd for C₂₅ H₂₉ N₃ O₄ Si (M⁺) 463.1927, found463.1941 LRMS (EI) m/z 463 (M⁺), 434, 419, 404, 390, 362, 346, 332, 167,131, 104, 91, 73, 57.

EXAMPLE 23

(20S)-7-[(2-Trimethylsilyl)ethyl]camptothecin (36c) (DB-172)

Following the procedure described in example 1-(2), a mixture of theiodopyridone (43) prepared in Example 21-(4) above (56.8 mg, 0.12 mmol)and phenylisonitrile (30.9 mg, 0.3 mmol) provided after flashchromatography (CH₂ Cl₂ /acetone 10:1) 28 mg (52%) of(20S)-7-[(2-trimethylsilyl)ethyl]camptothecin as a tan solid: [α]_(D) ²⁰+29.8 (c 0.2, CH₂ Cl₂); IR (CHCl₃, cm⁻¹) 2996, 2954, 1742, 1659, 1601,1557, 1250, 1158, 856; ¹ H NMR (300 MHz, CDCl₃) δ0.18 (s, 9H), 0.90-0.96(m, 2H), 1.04 (t, J=7.3 Hz, 3H), 1.86-1.95 (m, 2H), 3.07-3.13 (m, 2H),3.87 (s, 1H), 5.23. (s, 2H), 5.31 (d, J=16.3 Hz, 1H), 5.76 (d, J=16.3Hz, 1H), 7.64-7.69 (m, 2H), 7.80 (td, J₁ =8 Hz, J₂ =0.87 Hz, 1H), 8.03(d, J=8.3 Hz, 1H), 8.23 (d, J=8.3 Hz, 1H); ¹³ C NMR (75 MHz, CDCl₃)δ1.75, 7.95, 17.9, 24.2, 31.7, 49.4, 66.5, 72.9, 98.1, 118.5, 123.4,126.1, 126.7, 127.7, 130.3, 130.8, 147.1, 147.2, 149.6, 150.2, 152.0,157.8, 174.1; HRMS (EI) m/z calcd for C₂₅ H₂₈ N₂ O₄ (M⁺) 448.1818, found448.1819 LRMS (EI) m/z 448 (M⁺), 431, 374, 358, 311, 301, 208, 195, 165,149, 131, 118, 105, 93, 73.

Although the present invention has been described in detail inconnection with the above examples, it is to be understood that suchdetail is solely for that purpose and that variations can be made bythose skilled in the art without departing from the spirit of theinvention except as it may be limited by the following claims.

What is claimed is:
 1. A method of synthesizing compounds having theformula: ##STR33## via a 4+1 radical annulation/cyclization wherein theprecursor ##STR34## is reacted with an aryl isonitrile having theformula ##STR35## wherein R¹ and R² are independently the same ordifferent and are hydrogen, an alkyl group, an alkenyl group, an alkynylgroup, an alkoxy group, an aryloxy group, an acyloxy group,--OC(O)OR^(d), wherein R^(d) is an alkyl group, a carbamoyloxy group, ahalogen, a hydroxy group, a nitro group, a cyano group, an azido group,a formyl group, a hydrazino group, --C(O)R^(f) wherein R^(f) is an alkylgroup, an alkoxy group, an amino group or a hydroxy group, an aminogroup, --SR^(c), wherein R^(c) is hydrogen, --C(O)R^(f), an alkyl group,or an aryl group; or R¹ and R² together form a group of the formula--O(CH₂)_(n) O-- wherein n represents the integer 1 or 2;R³ is H, ahalogen atom, a nitro group, an amino group, a hydroxy group, or a cyanogroup; or R² and R³ together form a group of the formula --O(CH₂)_(n)O-- wherein n represents the integer 1 or 2; R⁴ is H, F, a C₁₋₃ alkylgroup, a C₂₋₃ alkenyl group, a C₂₋₃ alkynyl group, a trialkylsilyl groupor a C₁₋₃ alkoxy group; R⁵ is a C₁₋₁₀ alkyl group, an allyl group, abenzyl group or a propargyl group; R⁶, R⁷ and R⁸ are independently aC₁₋₁₀ alkyl group, a C₂₋₁₀ alkenyl group, a C₂₋₁₀ alkynyl group, an arylgroup or a --(CH₂)_(N) R⁹ group, wherein N is an integer within therange of 1 through 10 and R⁹ is a hydroxy group, alkoxy group, an aminogroup, an alkylamino group, a dialkylamino group, a halogen atom, acyano group or a nitro group; R¹¹ is an alkylene group, an alkenylenegroup, or and alkynylene group; R¹² is --CH═CH--CH₂ -- or --C.tbd.C--CH₂--; X is Cl, Br or I; and pharmaceutically acceptable salts thereof. 2.The method of claim 1, wherein R⁴ is H.
 3. The method of claim 1,wherein R¹ and R² are independently the same or different and are H, ahydroxy group, a halogen, an amino group, a nitro group, a cyano group,a C₁₋₃ alkyl group, a C₂₋₃ alkenyl group, a C₂₋₃ alkynyl group or a C₁₋₃alkoxy group.
 4. The method of claim 1, wherein R¹ and R² areindependently the same or different and are H, a C₁₋₃ perhaloalkylgroup, a C₁₋₃ aminoalkyl group, a C₁₋₃ alkylamino group or a C₁₋₃dialkylamino group.
 5. The method of claim 1, wherein R¹ and R² areindependently the same or different and are H, a methyl group, an aminogroup, a nitro group, a cyano group, or a hydroxy group.
 6. The methodof claim 1, wherein R¹ and R² are independently the same or differentand are H, a methylamino group, a dimethylamino group, an ethylaminogroup, a diethylamino group, a hydroxymethyl group, an aminomethylgroup, a methylaminomethyl group, or a dimethylaminomethyl group.
 7. Themethod of claim 1, wherein R³ is F, an amino group, or a hydroxy group.8. The method of claim 1, wherein R⁵ is an ethyl group.
 9. The method ofclaim 1, wherein R⁶, R⁷ and R⁸ are independently the same or differentand are a C₁₋₆ alkyl group, a phenyl group or a --(CH₂)_(N) R⁹ group,wherein N is an integer within the range of 1 through 6 and R⁹ is ahydroxy group, alkoxy group an amino group, an alkylamino group adialkylamino group, a halogen atom, a cyano group or a nitro group. 10.The method of claim 1, wherein R⁶, R⁷ and R⁸ are methyl groups.
 11. Themethod of claim 1, wherein R² and R³ form a methylenedioxy group, or a1,2-ethylenedioxy group.
 12. The method of claim 1, wherein R² is OH.13. The method of claim 1, wherein R² is NH₂.
 14. The method of claim 1,wherein R¹¹ is a C₁ -C₁₀ alkylene group, a C₂ -C₁₀ alkenylene group or aC₂ -C₁₀ alkynylene group.
 15. The method of claim 14, wherein R¹¹ is aC₁ -C₆ alkylene group, a C₂ -C₆ alkenylene group or a C₂ -C₆ alkynylenegroup.
 16. The method of claim 14, wherein R¹¹ is (CH₂)_(m) wherein m isan integer of 1 to
 6. 17. The method of claim 1 wherein X is Br or I.